Metal and Metal Oxide Nanoparticles To Enhance The Performance of Enzyme-Linked Immunosorbent Assay (ELISA) (ACS Applied Nano Materials) (2019)

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Review
Metal and Metal Oxide Nanoparticles to Enhance the
Performance of Enzyme-Linked Immunosorbent Assay (ELISA)
Yuan Gao, Yingzhu Zhou, and Rona Chandrawati
ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b02003 • Publication Date (Web): 09 Dec 2019
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Page 1 of 86 ACS Applied Nano Materials
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Metal and Metal Oxide Nanoparticles to Enhance
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the Performance of Enzyme-Linked Immunosorbent
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Assay (ELISA)
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21 Yuan Gao, Yingzhu Zhou and Rona Chandrawati*
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24 School of Chemical Engineering and Australian Centre for Nanomedicine (ACN), The
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27 University of New South Wales (UNSW Sydney), Sydney, NSW 2052, Australia.
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KEYWORDS. Nanoparticle, Metal, Metal Oxide, ELISA, Nanozyme, Enzyme, Antibody
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ACS Applied Nano Materials Page 2 of 86
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ABSTRACT
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7 Enzyme-linked immunosorbent assay (ELISA) is a technique designed for the detection
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10 and quantification of (bio)molecules in a liquid sample. It is a powerful tool in clinical
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diagnostics, food safety and environmental monitoring. However, the main limitation
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15 of conventional ELISA is its low sensitivity, which cannot meet the demand of analyte
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18 analysis in complex (biological) samples. Several successful approaches capitalizing on
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21 the unique physical and chemical properties of nanoparticles to improve the
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23 performance of traditional ELISA have been reported. In this review, we aim to
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26 demonstrate diverse strategies designed to date that use metal and metal oxide
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29 nanoparticles to overcome challenges associated with ELISA sensitivity and stability. In
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particular, we discuss metal and metal oxide nanoparticles as carriers to load enzymes
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34 and antibodies for signal amplification, as enzyme mimics to replace the natural
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37 enzyme label, and as signal transducers to provide fluorescence or luminescence signals
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as an alternative output.
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4 Introduction
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8 Enzyme-linked immunosorbent assay (ELISA) has been widely used for the
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10 detection and quantification of antigens, antibodies, peptides, proteins, hormones, and
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13 other molecules, with applications ranging from clinical diagnostics, food safety to
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16 environmental monitoring.1-5 Compared with other detection techniques such as liquid
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chromatography, gas chromatography, and mass spectrometry, ELISA represents a
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21 simple, reliable, and sensitive analytical tool for rapid screening of target analytes. The
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24 essential components of conventional ELISA include: i) an antigen, ii) antibodies
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towards this antigen, iii) an enzyme label, and iv) a chromogenic substrate, which
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29 yields a measurable color product upon reaction with the enzyme label.
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33 There are four major types of ELISA: direct, indirect, sandwich, and competitive
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36 assays (Figure 1). In direct ELISA, the analyte to be tested is adhered on a well plate,
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38 which then complexes with an enzyme-labeled primary antibody. After antigen
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41 immobilization and before incubation with an antigen-specific antibody, a blocking
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44 agent (typically bovine serum albumin (BSA)) is added to saturate available sites and to
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46 prevent non-specific antibody binding. This is done to maximize the signal-to-noise
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49 ratio. Once the antibody binds to the antigen (analyte), the enzyme catalyzes a
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52 chromogenic substrate, resulting in a visible colorimetric output that is measured by a
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UV-Vis spectrophotometer. The concentration of the analyte is directly proportional to
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the color intensity. In indirect ELISA, primary antibodies are not labeled to retain their
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6 maximum immunoreactivity. Instead, enzymes are conjugated onto secondary
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9 antibodies that recognize the primary antibodies. In sandwich ELISA, capture
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antibodies are first coated on a well plate, followed by the addition of the sample or
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14 analyte to be tested, with the rest of the procedure following the indirect ELISA
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17 method. Sandwich ELISA usually gives a higher signal-to-noise ratio, but requires more
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20 optimization compared to direct and indirect ELISA. Due to its better sensitivity,
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22 specificity, and flexibility, sandwich ELISA is the most commonly used assay method.
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25 Competitive ELISA method is more complex since it involves a reference antigen. The
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28 reference antigen is pre-coated on a well plate. Then, the sample to be tested is
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incubated with enzyme-labeled antibodies before adding to the wells. Depending on
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33 the amount of antigen in the sample, varying amounts of free antibodies will be
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36 available to bind to the reference antigen. Therefore, the more antigen there is in the
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sample, the weaker the signal. In this case, the concentration of the antigen in the
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41 sample is inversely proportional to the color intensity. The crucial factors that affect
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44 ELISA signal generation include the specificity and affinity of the antibody to the
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47 antigen, the concentration and biocatalytic activity of the enzyme, and the choice of
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49 substrates and signal detection.
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41 Figure 1. a) Overview of direct, indirect, sandwich, and competitive ELISA. b)
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43 Schematic illustration of the roles of nanoparticles (NPs) in improving the performance
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46 of traditional ELISA by acting as: (i) carriers to load enzymes and antibodies for signal
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49 amplification, (ii) enzyme mimics to replace natural enzyme labels, and (iii) signal
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transducers to provide fluorescence or luminescence signals as an alternative output.
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7 Colorimetric ELISA is a commonly used and favored detection technique because it
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10 is simple and can easily be observed by the naked eye.6 Amongst the colorimetric signal
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generating formats, horseradish peroxidase (HRP)/3,3',5,5'-tetramethylbenzidine (TMB)
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15 pair is the preferred option. TMB is a highly sensitive substrate that yields a soluble
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18 blue product measurable at 650 nm upon oxidation by HRP in the presence of hydrogen
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21 peroxide (H2O2). It is an ideal substrate for kinetic studies due to its fast reaction rate.
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23 TMB can also be used in endpoint assays by stopping the enzymatic reaction with the
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26 addition of acid (e.g. HCl or H2SO4). A yellow reaction product is then formed, and the
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29 absorbance can be monitored at 450 nm. Other colorimetric substrates for HRP include
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2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and o-
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34 phenylenediamine (OPD), however they are less sensitive than TMB. In addition to
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37 HRP (Mw 44 kDa), alkaline phosphatase (ALP, Mw 140 kDa) is used extensively in
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ELISA.7 HRP is preferred as it is cheaper, more stable, and has a faster catalytic rate.
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42 Furthermore, due to its small size, HRP does not cause steric hindrance with antigen-
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45 antibody complexes. However, HRP is incompatible with preservatives found in many
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48 biological buffer solutions, as they inactivate peroxidase activity. One drawback of
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50 colorimetric assays is the limited dynamic range imposed due to the upper limit of the
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53 optical density (OD). To overcome this, alternative HRP substrates including Amplex
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56 Red, QuantaRed, and QuantaBlu are available, which produce a fluorescence signal
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when detecting the target analyte. Fluorescence detection is more sensitive than
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6 colorimetric-based assays and can widen the dynamic range.
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10 Although there are many ready-to-use commercially available ELISA kits, this
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technique is not without limitations. The basic nature of an ELISA limits a single assay
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15 to the detection of a single target analyte. ELISA has low sensitivity, which cannot meet
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18 the demand of biomarker analysis in complex biological samples. The enzyme label,
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21 being a natural protein, can lose its catalytic activity due to heat-, pH-, or chemical-
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23 induced denaturation.8-9 Therefore, transport and storage conditions need to be strictly
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26 controlled. Furthermore, colored samples could also interfere with the colorimetric
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29 output.
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To improve the performance of traditional ELISA, several strategies have been
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35 developed which can be broadly categorized into non-nanotechnnology-based and
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38 nanotechnology-based. For the former, contributions include more sensitive
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chromogenic and fluorogenic substrates for HRP,10-11 blocking agents and coatings with
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43 higher efficacy to minimize background noise,12-14 improving antibody affinity,15-17
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46 novel antibody fragments and nanobodies,18-19 biotin-streptavidin systems,20-21 and
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49 ELISA coupled with polymerase chain reaction22 or microfluidics.23-24
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52 The convergence of nanotechnology and ELISA has led to remarkable
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55 improvements in assay sensitivity and ease of operation. Noble metal nanoparticles
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(e.g. gold and silver) exhibit unique optical properties that arise from the Localized
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6 Surface Plasmon Resonance (LSPR) phenomenon. The spectral position of the LSPR
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9 strongly depends on nanoparticle size, shape, composition, and interparticle spacing.25-
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26 Therefore, a minor change in the local environment of metal nanoparticles (e.g. due to
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14 the presence of analytes) leads to modulation in their LSPR peak and changes of
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17 nanoparticle solution color that can be determined quantitatively and qualitatively. de
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20 la Rica and Stevens conceptualized the idea of gold nanoparticle (AuNP) synthesis and
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22 growth kinetics as a means to generate colorimetric ELISA signal, and they developed
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25 plasmonic ELISA for the ultrasensitive detection of prostate specific antigen and HIV-1
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28 capsid antigen p24 in patient serum samples.27 In contrast to traditional ELISA where
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enzyme catalysis generated a colored product, the developed plasmonic sandwich
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33 ELISA generated colored nanoparticle solutions through activity of catalase tagged on
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36 secondary antibodies. In the absence or presence of target analytes, growth kinetics of
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AuNPs was tuned resulting in a red or blue color solution, respectively. Many
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41 variations of plasmonic ELISA have been developed, with colorimetric signal
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44 generation based on induced aggregation of nanoparticles28-32 or transformation in
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47 nanoparticle shape.33-34 Several excellent reviews have been recently published
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49 documenting work related to plasmonic ELISA35-38 as well as Surface-Enhanced Raman
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52 Scattering-based ELISA.39-41
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In this review, we focus on approaches to improve traditional ELISA capitalizing on
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6 the unique physical and chemical properties of metal and metal oxide nanoparticles. In
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9 particular, we discuss three functions of nanoparticles: i) nanoparticles as carriers to
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load enzymes and antibodies for signal amplification, ii) nanoparticles with enzyme-
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14 mimicking activity to replace the natural enzyme labels, and iii) nanoparticles as signal
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17 transducers to provide fluorescence or luminescence signals as an alternative output
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20 (Table 1). A range of nanoparticle-antibody or nanoparticle-enzyme functionalization
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25 activity and assay sensitivity are discussed.
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33 Nanoparticles as carriers
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In conventional ELISA, one of the key contributors limiting assay sensitivity is the low
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39 enzyme to antibody ratio, typically 1:17, 42 (Figure 1a), resulting in a poor signal-to-noise ratio.
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41 To enhance the assay sensitivity, a possible solution is to increase the number of enzyme
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43 molecules in the final antigen-antibody-enzyme complex. This will increase the catalysis of the
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46 substrate and lead to signal amplification in a single recognition reaction. To achieve this,
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48 various metal and metal oxide nanoparticles have been utilized as carriers for multiple enzymes
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50 and antibodies.43-44 Nanoparticles are excellent candidates for this purpose due to their large
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53 surface area-to-volume ratio, high loading capacity, facile synthesis, chemical stability, and ease
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55 of bioconjugation.45-46 The capability of immobilizing multiple enzymes and antibodies
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on the surface of a single nanoparticle can significantly enhance ELISA sensitivity and
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6 widen the detection range without a complex synthesis procedure.43, 47 Currently, the
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9 most common strategies to functionalize enzymes and antibodies onto nanoparticles are through
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14 enzymes on nanoparticles, it is important to balance the amount because too much can
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lead to high background signal, resulting in a poor signal-to-noise ratio.
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AuNPs
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27 AuNPs have been widely employed in biosensing and analytical applications48-52
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30 due to their unique optical and catalytic property, stability, and surface functionality,
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33 which provide a versatile platform for conjugation with antibodies,53-54 peptides,55-56
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35 aptamers,57-59 and other recognition elements. Compared to the traditional ELISA
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38 technique, ELISA coupled with AuNPs has been shown to improve assay sensitivity,
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41 lower limit of detection (LOD), and shorten assay time.43
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Merkoçi and co-workers developed a direct sandwich ELISA for the detection of
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47 CA15-3 breast cancer biomarker and used AuNPs as carriers of anti-CA15-3-HRP
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50 antibody (Figure 2a).43 Capture anti-CA15-3 antibody was first immobilized on a well
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plate. In the presence of CA15-3, the antigen bound to the capture antibody and
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55 complexed with AuNP-anti-CA15-3-HRP. TMB was then added to the well, followed by
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the addition of sulfuric acid to stop the color development, and the absorbance was
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6 measured at 450 nm. The AuNP-ELISA assay could detect a biologically relevant level
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9 of CA15-3 between 0 – 250 U/mL, with a linear range of 0 – 60 U/mL (Figure 2b). Using
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AuNPs as enhancers resulted in a LOD of 0.012 OD·mL/U, double the sensitivity
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14 without the use of AuNPs. The incubation time with TMB also significantly reduced
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17 from 30 min on a classical ELISA to only 5 min. The higher sensitivity and faster
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20 response time are due to the attachment of a multiple enzyme system on the
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22 nanoparticle (19 enzyme-labeled antibodies per 15 nm-diameter AuNP determined via
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25 gold aggregation test), which generated an amplified optical signal. AuNP-anti-CA15-3-
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28 HRP conjugates were prepared by the direct adsorption of anti-CA15-3-HRP onto
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AuNPs for 30 mins at pH 7. They can be stored for a week at 4 °C without losing
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33 bioactivity. To achieve high signal-to-noise ratio, optimization of AuNP-antibody
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36 concentration to be used in the ELISA test is required. Undiluted high-concentration
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AuNP-anti-CA15-3-HRP resulted in a low signal-to-noise ratio. On the other hand,
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41 when a 1:100 diluted AuNP-anti-CA15-3-HRP complex was used, very low signals were
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44 recorded even at high concentration of CA15-3. Therefore, a good balance between low
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47 non-specific signal and high sensitivity has to be considered in optimization of the
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49 assay conditions.
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39 Figure 2. a) Schematic illustration of a direct sandwich ELISA for the detection of CA15-
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42 3 breast cancer biomarker with and without AuNPs as carriers for anti-CA15-3-HRP
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45 antibodies. b) ELISA test for CA15-3 performed in the presence (red) and absence (blue)
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of AuNPs. Reproduced with permission from ref 43. Copyright 2010 American Chemical
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50 Society.
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AuNP-antibody functionalization methods influence antibody activity and assay
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6 sensitivity. To identify an optimized approach, Ciaurriz et al. compared four antibody
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9 functionalization strategies towards enhancing ELISA performance: i) direct adsorption
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of antibodies to AuNPs via electrostatic and hydrophobic interactions, ii) directional
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14 conjugation using a hetero-bifunctional linker (e.g. hydrazide-polyethylene glycol-
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17 dithiol), iii) covalent conjugation using a polyethylene glycol linker and EDC/NHS
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20 carbodiimide method, and iv) a combination of directional and adsorption strategies
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22 (Figure 3a).47 The modified AuNP probes were assessed in model rabbit IgG and anti-
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25 rabbit IgG ELISA and the colorimetric response was compared. In theory, directional
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28 conjugation is expected to have better efficiency because fragment antigen-binding
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(Fab) portions of the antibody are directed outward from the AuNP surface and
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33 therefore, more antigen-binding sites will be available.47 Interestingly, the direct
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36 adsorption method showed the highest signal-to-noise ratio compared to the other three
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strategies (Figure 3b). The authors did not report the number of antibody molecules
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41 bound to AuNPs, however they postulated that a direct adsorption method led to the
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44 formation of protein multilayers and therefore the total number of antibodies bound to
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47 AuNPs was higher. The authors then applied this direct adsorption method and
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49 developed AuNP-ELISA for the detection of gliadin from wheat gluten. The AuNP-
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52 ELISA resulted in a LOD of 180 pg/mL gliadin, more than seven times higher sensitivity
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55 than a classical ELISA.47 Although the direct adsorption method could lead to higher
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antibody surface coverage on nanoparticles (compared with directional or covalent
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6 conjugation binding), this method leads to random orientation of the antibody. A
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9 previous study has reported only 23  6% of the directly adsorbed antibodies
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maintained antigen-binding activity.60
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49 Figure 3. a) Schematic illustration of four different AuNP-antibody functionalization
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52 methods: (a) direct adsorption, (b) directional conjugation using a hetero-bifunctional
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linker, (c) covalent conjugation through antibody and enzyme amine groups, (d) a
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combination of directional and adsorption strategies. b) Results of ELISA comparing
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6 AuNP probes prepared by adsorption, covalent, or directional and adsorption methods.
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9 Reproduced with permission from ref 47. Copyright 2017 Beilstein-Institut.
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16 In addition to functionalization methods, Kim et al. studied the effect of AuNP size
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19 in ELISA performance.61 In this study, HRP-labeled anti-C reactive protein antibodies
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22 (anti-CRP-HRP) were conjugated onto AuNPs of varying diameters (5 nm, 10 nm, 15
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24 nm, 20 nm, 30 nm, and 50 nm) via direct adsorption method. The number of anti-CRP-
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27 HRP immobilized on the nanoparticles was determined to be 3, 13, 32, 66, 102, and 142
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30 for AuNPs of 5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm, respectively. Although the
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number of anti-CRP-HRP increased, ELISA with AuNPs of 5 nm diameter produced the
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35 highest colorimetric response. It was suggested that there was a synergistic effect
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38 between particle size and number of antibodies conjugated. This observation
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corresponds with findings by Tripathi and Driskell, who reported that higher antibody
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43 surface coverage did not necessarily mean higher antigen-binding activity.60 The AuNP-
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46 ELISA system enabled the detection of 0.1 ng/mL of CRP within 30 seconds, in contrast
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49 to 10 ng/mL for the ELISA run in the absence of AuNP with 30 min incubation.61
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52 The improvement offered by an AuNP-ELISA system was also demonstrated for
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55 the detection of respiratory syncytial virus,62 procalcitonin (sepsis biomarker),63
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fumonisins (mycotoxins),64 and cancer cells expressing high levels of epidermal growth
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6 factor receptor (EGFR).65 Billingsley et al. coated antibodies specific to EGFR on 150 nm-
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9 diameter gold nanoshells, which were composed of a 120 nm silica core and a 15 nm
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thick gold shell (Figure 4).65 Each nanoshell has an average of 50 anti-EGFR antibodies
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14 attached on the surface. The authors found that ELISAs performed with unconjugated
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17 anti-EGFR antibodies required 25× dilution of the antibody to be effective in detecting
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20 EGFR-expressing cells. Using AuNP-anti-EGFR, the assay worked well at 1000×
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22 antibody dilution. The AuNP-based system significantly reduced the number of
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25 antibodies required (40-fold), thereby lowering the cost of the assay.
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51 Figure 4. Schematic illustration of nanoparticle-modified ELISA for the detection of
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53 epidermal growth factor receptor (EGFR)-expressing cancer cells. NS: nanoshell.
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56 Reproduced with permission from ref 65. Copyright 2017 PLOS One.
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7 Wang et al. further developed AuNP-ELISA with a multiple AuNP system.66 In
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10 their study, polyamidoamine (PAMAM) dendrimers were used to assemble 14 AuNPs
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together to form a necklace-like circle, which enabled the immobilization of 19-20 HRP
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15 on the AuNP-dendrimer surface. The assembled AuNPs provided more binding sites
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18 for enzymes compared with a single AuNP, which could immobilize only 11-12 HRP.
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21 The AuNP-dendrimer-ELISA system successfully detected human chorionic
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23 gonadotropin at a linear range of 0.1 - 6.4 IU/L, with a LOD of 0.03 IU/L, 20 times lower
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26 compared to single AuNP-ELISA system and 27 times lower than the traditional ELISA.
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Chen and co-workers developed an alternative approach where they fabricated
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36 gold nanoparticle layers (GNPLs) on commercial ELISA plates with the aim to improve
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39 the binding of capture antibodies to the surface.67-68 They showed that the activity of the
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antibody immobilized on the GNPL surface was 108% higher than when adsorbed on
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44 an unmodified plate. GNPL modification led to superhydrophilic surfaces, which
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47 improved binding efficiency of the antibody while maintaining antibody activity. The
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50 GNPL-modified ELISA significantly improved the LOD by 125-fold, from 9.6 ng of
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52 antithrombin using traditional ELISA to 76.8 pg.67
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Magnetic nanoparticles (MNPs)
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7 MNPs based on iron oxides have been integrated into ELISA system for the
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10 detection of a variety of target analytes including proteins, bacteria, viruses,
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mycotoxins, and allergens.4, 63, 69-72 Similar to the AuNP-ELISA system, MNPs are
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15 typically conjugated with enzyme-labeled antibodies. Due to the unique properties of
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18 MNPs, a magnetic field can be applied after immunochemical reactions to enable a
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21 rapid separation of reactants. Magnetic separation also allows concentration of analytes
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23 from a large volume of the original sample into a smaller volume for analysis.
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27 Speroni et al. developed a MNP-dendrimer-ELISA system for the sensitive detection
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30 of Ara h3/4 peanut allergens.44 Similar to the work reported by Wang and co-workers,66
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PAMAM dendrimers were used in this study. Amine-functionalized MNPs were first
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35 conjugated with PAMAM-sodium carboxylate dendrimers via the EDC/NHS reaction.
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38 Following this, antibodies were covalently attached to the MNP-dendrimers via the
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same reaction mechanism. Using MNP-dendrimers, a three-fold enhancement of the
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43 assay sensitivity was achieved, with a LOD of 0.2 mg peanuts/kg food matrix.
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46 Compared to traditional ELISA, MNP-dendrimer-antibodies with the captured
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49 allergens can be easily harvested on a magnet, separated from the solutions, and
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51 washed before quantification.
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Petrakova et al. also demonstrated the higher sensitivity and faster response time of
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6 analyte detection with the use of MNPs, both as carriers of antibodies and as a means of
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9 preconcentrating antigens, in a competitive ELISA format.73 Aflatoxin B1 (AFB1) is a
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highly toxic mycotoxin produced by various species of the mold Aspergillus. This toxin
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14 is widely spread in nature and can severely contaminate food supplies of human and
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17 animals.74 In this study, 10 nm-diameter MNPs were conjugated with anti-AFB1 via the
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20 direct adsorption method, which led to 24% immobilization yield of antibodies. The
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22 MNP-antibody-AFB1 complexes were preconcentrated 50-fold by precipitation with a
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25 magnetic field and then HRP-labeled AFB1 was added into the reaction, followed by
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28 addition of substrates (Figure 5a). The higher the AFB1 content of the sample, the lower
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the binding of the HRP-labeled AFB1. Using MNPs, a 10-fold lower LOD was achieved
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33 (2 pg/mL, Figure 5b) and the total assay duration was reduced from 120 min to 50 min.
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36 Although integrating MNPs in ELISA can significantly improve assay sensitivity, they
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tend to aggregate due to magnetic dipole-dipole attraction between particles.75
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25 Figure 5. a) Schematic illustration of a competitive MNP-ELISA system for the detection
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28 of aflatoxin B1 (AFB1): (1) AFB1-containing sample, (2) MNP conjugates with anti-AFB1
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antibody, (3) magnet, (4) enzyme-labeled AFB1, (5) enzyme substrate. b) Comparison of
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33 limit of detection and working range of different ELISA versions for AFB1. Reproduced
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36 with permission from ref 73. Copyright 2015 Royal Society of Chemistry.
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43 Paper is an attractive material as a supporting matrix in sensing applications due to
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46 its low-cost, portability, disposability, biodegradability, and lightweight feature.76-79
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49 Moreover, paper can be easily coated with biomolecules, whilst its fiber network
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51 increases surface area and loading capacity. Reguera and co-workers reported a
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54 magnetic paper-based ELISA for the detection of IgM-dengue antibodies with two
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orders of magnitude higher sensitivity than traditional ELISA.80 In their work, core-
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6 shell magnetite-polydopamine nanoparticles were used as a carrier for anti-IgM
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9 antibodies, with two fabrication processes: i) Fe3O4 MNPs were first coated on paper,
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followed by growing polydopamine shell, and finally conjugating with anti-IgM
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14 antibodies or ii) polydopamine-modified Fe3O4 MNPs were first synthesized prior to
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17 deposition on a paper surface, followed by anti-IgM antibody conjugation (Figure 6a).
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20 In this study, polydopamine served as a linker for antibody conjugation, where
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22 antibody amino groups reacted with polydopamine quinone groups via Michael-type
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25 addition. The authors found that method (i) led to higher antibody coupling efficiency
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28 compared to method (ii), possibly due to fewer amounts of magnetite-polydopamine
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per area for the latter. The optimized system was then tested for dengue recognition,
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33 which showed good linearity in a range of 0.03 to 1 μg/mL, with 700 times lower LOD
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36 in PBS and 500 times lower LOD in human serum than traditional ELISA or MNP-
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ELISA without depositing on papers (Figure 6b).
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35 Figure 6. a) Schematic illustration of a magnetic paper-based ELISA for the detection of
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38 IgM-dengue antibodies. b) The analytical performance of magnetic paper-based ELISA
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40 compared with traditional ELISA or magnetic nanoparticle-ELISA without depositing
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43 on paper. Reproduced with permission from ref 80. Copyright 2017 Royal Society of
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46 Chemistry.
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53 Silica nanoparticles (SNPs)
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In contrast to previous examples, where nanoparticles are used solely to provide
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6 more binding sites for enzyme-labeled antibodies in an ELISA system, Lei et al.
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9 synthesized dendritic mesoporous SNPs both as a carrier for antibody and a reservoir
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for enzyme loading.81 The mesoporous SNPs had a large surface area (484 m2/g) and
13
14 pore size (14.5 nm), which enabled loading of 390 mg HRP/g particles. HRP was loaded
15
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17 to the SNPs via glutaraldehyde crosslinking. Glutaraldehyde is one of the most widely
18
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20 used reagents for enzyme crosslinking.82-83 Although there are reports documenting
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22 that glutaraldehyde could cause protein denaturation, the authors showed that this
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25 immobilization method retained 67% of the enzyme activity. Using the SNP-ELISA
26
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28 system, an ultrasensitive assay with 2000 times enhancement of sensitivity for insulin
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detection was demonstrated (LOD = 3.85 fg/mL in human serum). However, the authors
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33 reported that trace amounts of SNPs could stick on the wells and produce non-specific
34
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36 signals.
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In addition to HRP, other groups have reported the use of mesoporous SNPs for
41
42 loading of other classes of enzyme, such as glucose oxidase84-86 and catalase.87-88 Kim
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45 and co-workers encapsulated glucose oxidase into mesoporous SNPs and showed that
46
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48 enzyme loading via glutaraldehyde treatment retained 98% of the enzyme activity,
49
50 compared to the physical adsorption or covalent attachment method, which retained
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53 only 20% or 49% of the enzyme activity respectively.86 Anti-human IgG antibodies (anti-
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56 hIgG) were then conjugated onto the enzyme-loaded SNPs by reduction amination
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coupling, and hIgG was detected in a sandwich assay format (Figure 7a). SNP-ELISA
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6 led to a LOD of 0.5 ng/mL (3.3 pM) hIgG, while the LOD of conventional ELISA was 10
7
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9 ng/mL (67 pM) hIgG (Figure 7b). This represents 20 times higher sensitivity compared
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to the conventional immunoassay. Particle shape also plays an important role in
13
14 influencing ELISA sensitivity. SNP-ELISA with spherical mesoporous SNPs is 20-fold
15
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17 more sensitive than the same system using amorphous mesoporous SNPs.86 This is
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20 because antibodies conjugated on concave surfaces were not accessible due to the steric
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22 hindrance against their binding to the target analyte.
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Figure 7. a) Schematic illustration of a sandwich SNP-ELISA for the detection of hIgG.
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45 Mesoporous SNPs are used both as a carrier for antibody and a reservoir for enzyme
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48 (glucose oxidase, GO) loading. b) Calibration curves of the sandwich immunoassay for
49
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51 the detection of hIgG at various concentrations from 0.1 to 10000 ng/mL using antibody-
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53 conjugated GO (conventional immunoassay) and antibody-conjugated spherical GO-
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loaded SNPs. (SNP-ELISA). NER: nanoscale enzyme reactor. MPS: mesoporous silica.
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6 Reproduced with permission from ref 86. Copyright 2014 Royal Society of Chemistry.
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14 Nanoparticles as enzyme mimics
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18 Enzyme label is an important component in ELISA and its catalytic activity dictates
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assay sensitivity. Although naturally occurring enzymes are efficient and specific
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23 catalyst proteins, they often suffer from intrinsic shortcomings such as low stability and
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26 short shelf life.89 Furthermore, enzymes cannot withstand harsh conditions, such as heat
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29
or pH changes.35, 38 To overcome these limitations, efforts have been devoted to the
30
31 exploration of nanomaterials as enzyme mimics or nanozymes.89-93 Enzyme mimics are
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34 attractive substitutes for their natural counterparts in ELISA because they are highly
35
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37 stable, can be mass-produced at relatively low cost, and allow facile modification with
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39 biomolecules (e.g. antibody). With relevance to ELISA, work has focused on enzyme
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42 mimics with peroxidase-like activity.
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49 AuNPs
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53 Many noble metal nanoparticles, including AuNPs, have been reported as
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56 peroxidase mimics and are widely used as a component in sensors.94-97 AuNPs catalyze
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the oxidation of TMB in the presence of H2O2 to generate a blue product, mimicking the
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6 function of HRP. Superficial gold atom is a contributing factor to the observed
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9 peroxidase-like activity.
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Wang et al. applied the peroxidase-like activity of AuNPs in ELISA for the detection
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15 of human IgG (h-IgG), which is an indicator for renal and nervous system
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18 dysfunction.98 In this case, a direct sandwich ELISA was developed, and anti-IgG-AuNP
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21 was used instead of anti-IgG-HRP. In the presence of h-IgG, the antigen bound to the
22
23 anti-IgG capture antibody and formed complexes with anti-IgG-AuNP. Surface
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26 modification is crucial in preventing the aggregation of colloidal nanoparticles,
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29 however, previous studies have shown that unmodified AuNPs have a significantly
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higher catalytic activity to peroxidase substrates compared with modified or capped
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34 AuNPs.99-100 Therefore, to improve the catalytic ability of anti-IgG-AuNP, gold growth
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37 solution (HAuCl4.4H2O and NH2OH.HCl) was added in the ELISA system with the aim
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to deposit gold on the AuNPs and to recover the peroxidase-like activity. Following
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42 this, TMB and H2O2 were added, and the colorimetric response was monitored at 652
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45 nm within 15 min incubation. Due to the gold enhancement, the AuNP-ELISA assay
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48 could detect h-IgG in the range from 0.7 to 100 ng/mL, with a LOD of 0.3 ng/mL, which
49
50 is lower compared to ELISA using anti-IgG-HRP (5.0 to 200 ng/mL with LOD of 1.6
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53 ng/mL).98 When the colorimetric output was followed by the naked eye readout, as low
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56 as 5 ng/mL of h-IgG could be detected.
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Nanoparticle size plays a key role in tuning assay sensitivity, with smaller AuNPs
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6 (e.g. 14 nm) having higher catalytic activity towards TMB compared to larger AuNPs
7
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9 (e.g. 100 nm) due to the larger surface area-to-volume ratio.101 Similar to HRP, AuNPs
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can also catalyze the conversion of ABTS into colored products. ABTS is negatively
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14 charged, while TMB is positively charged. Therefore, depending on the capping of the
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17 AuNPs, one substrate may be preferred over another. For example, Liu et al. showed
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20 that cysteamine-capped AuNPs possessed stronger affinity to ABTS than TMB, leading
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22 to higher catalytic activity with the former substrate.101 AuNP-based immunosensing
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25 strategy with 4-nitrophenol as the substrate for AuNP catalysts has also been
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28 reported.102
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To further improve the catalytic efficiency and performance of AuNP-ELISA, Park
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34 and co-workers developed hybrid structures of AuNPs and graphene (Grp-AuNPs) to
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37 combine the peroxidase-like activity of the individual component.103 Grp-AuNPs were
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synthesized by mixing graphene flakes with HAuCl4 and HCOONa, and then anti-
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42 norovirus (anti-NoV) antibodies were conjugated to Grp-AuNPs via covalent
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45 conjugation method (Figure 8). Using the hybrid Grp-AuNP-anti-NoV, norovirus-like
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48 particles (NoV-LPs) could be detected in a linear range of 100 pg/mL to 10 μg/mL with a
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50 LOD of 92.7 pg/mL. The sensitivity of this assay is 112 times greater compared with
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53 conventional ELISA and 41 times greater compared with commercial diagnostic kits.
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22 Figure 8. Schematic illustration of a direct Grp-AuNP-ELISA for the detection of
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norovirus-like particles (NoV-LPs). Grp-AuNP structures are synthesized in one step
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27 using graphene flakes, HAuCl4, and HCOONa. ELISA test for NoV-LPs performed
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30 using Grp-AuNP-anti-NoV (red) compared with conventional ELISA (black).
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Reproduced with permission from ref 103. Copyright 2016 Elsevier.
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40 MNPs
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44 The peroxidase-like activity of Fe3O4 and Fe2O3 MNPs is dependent on size, pH,
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46 temperature, and H2O2.104 The smaller the size, the higher the catalytic activity due to
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49 the high surface area-to-volume ratio. Compared with HRP, Fe3O4 MNPs show
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52 peroxidase activity for a larger pH range (0-12) with improved temperature tolerance
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(4-90°C).105 However, MNPs require H2O2 concentrations two orders of magnitude
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higher than HRP to reach the maximum level of peroxidase activity.104 Fe3O4 NPs
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6 catalyze a wide range of substrates, including TMB,106 ABTS,107 OPD,104 and 3,3'-
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9 diaminobenzidine (DAB).108
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In contrast with AuNPs,99-100 surface modification of MNPs does not affect the
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15 peroxidase-like activity of the nanoparticles. Yang et al. showed that amine-
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17
18 functionalized Fe3O4 MNPs exhibited the same catalytic efficiency as pristine MNPs.
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21 More importantly, the modified MNPs were successfully applied in a sandwich ELISA
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23 for the detection of human chorionic gonadotropin (hCG).109 After eight rounds of
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26 recycling reactions with TMB, the Fe3O4 MNPs retained 50-60% of their catalytic
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29 activity.
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32
Similar to the concept of Grp-AuNP-ELISA mentioned previously103 (i.e. combining
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35 the peroxidase-like activity of the individual component), Liu et al. developed ZnFe2O4-
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38 multiwalled carbon nanotube (ZnFe2O4@MWNT)-ELISA110 to combine the peroxidase
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catalytic performance of ZnFe2O4 MNPs111 and MWNTs.112 A one-step hydrothermal
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43 approach was used to fabricate ZnFe2O4@MWNTs, using ZnCl2, FeCl3, and MWNTs.
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46 ZnFe2O4@MWNTs were then functionalized with anti-carcinoembryonic antigen (anti-
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49 CEA) antibodies using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde as
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51 coupling agents. A sandwich ELISA was developed and adapted to a paper format
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54 (Figure 9a). This method can detect CEA in human serum in a linear range of 0.005 to 30
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ng/mL with a LOD of 2.6 pg/mL (Figure 9b). The high sensitivity is attributed to the
4
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6 high peroxidase-like activity of the combined ZnFe2O4@MWNTs and their high affinity
7
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9 to interact with substrates. When stored at room temperature, the paper-based
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ZnFe2O4@MWNT-ELISA retained 96% of the initial response in the first 20 days and
13
14 50% in the next 40 days. Remarkable stability was shown upon storage in the fridge and
15
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17 freezer, with up to 96% of the initial response being retained after 60 days of storage.
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20 The peroxidase mimics offer a stability that cannot be achieved using natural HRP.
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41 Figure 9. a) Schematic illustration of a paper-based ZnFe2O4@MWNT-ELISA for the
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43 detection of carcinoembryonic antigen (CEA). Chitosan and porous gold are deposited
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46 onto a filter paper to entrap capture antibodies. ZnFe2O4@MWNTs are functionalized
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49 with anti-CEA antibodies using (3-aminopropyl)triethoxysilane (APTES) and
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52
glutaraldehyde (GA) as coupling agents. The immunosensor response is quantified as a
53
54 color change resulting from ZnFe2O4@MWNTs catalyzing the oxidation of TMB in the
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presence of H2O2. b) Calibration curve of the sandwich immunoassay for the detection
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6 of CEA at various concentrations. The photographs (top) indicate changes in color
7
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9 intensity in response to the increasing concentration of CEA. Reproduced with
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permission from ref 110. Copyright 2014 Royal Society of Chemistry.
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19 It is worth noting that while extensive studies of peroxidase-like Fe3O4 MNP have
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22 been reported, Gumpelmayer et al. recently reported a contradictory finding that Fe3O4
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24 MNPs do not possess peroxidase-like activity towards both chromogenic and
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27 fluorogenic substrates.113 The authors found that removing free iron ions from the
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30 surface of Fe3O4 nanoparticles (with a Chelex buffer) eliminated their intrinsic
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32
peroxidase-like activity. This suggests that the reported catalytic activity was likely
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34
35 assigned to metal impurities from commercially available nanoparticles, which may
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38 result in misinterpretation of data.
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45 Ceria (CeO2) nanoparticles
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49 Similar to AuNPs and MNPs, ceria can catalyze peroxidase substrates like TMB and
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52 ABTS to provide colorimetric responses.114-115 Ceria nanoparticles have also been
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54 reported to have multi-enzyme activity, including superoxide dismutase, catalase,
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phosphatase, and oxidase.116 The ratio of Ce3+/Ce4+ is a contributing factor to the
4
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6 enzyme-mimicking activity. Higher ratios on the surface of ceria nanoparticles results in
7
8
9 superoxide dismutase mimetic activity, while lower ratios demonstrate peroxidase and
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catalase mimetic activity.117 The Ce3+/Ce4+ ratio can be tuned by nanoparticle size, where
13
14 the concentration of Ce3+ increases with a decrease in particle size.118 CeO2 can catalyze
15
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17 the oxidation of TMB in the absence of H2O2. In fact, Guo et al. showed that H2O2 could
18
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20 be a promoter or an inhibitor to nanoceria’s peroxidase activity depending on H2O2
21
22 concentration.119 At micromolar concentrations, oxidation of H2O2 by Ce4+ was
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24
25 thermodynamically favorable and the surface of CeO2 was rich in Ce3+. As the Ce3+/Ce4+
26
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28 ratio increased, it led to inhibition of nanoceria peroxidase activity. On the other hand,
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at millimolar concentrations, the peroxidase activity of nanoceria was restored. This
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33 suggests the available amount of Ce3+ determines the reaction efficiency and catalytic
34
35
36 activity. The authors exploited the off-on control of peroxidase activity of nanoceria to
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develop a highly sensitive detection system for H2O2.119
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42 Qu and co-workers developed a sandwich ceria-ELISA for the detection of CA15-3
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45 breast cancer biomarkers.120 To increase the catalytic performance of ceria, they
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48 synthesized porous nanorods of ceria (PN-ceria) with a diameter of 8 nm and a length
49
50 of 60 nm, with a pore size of 2-4 nm. The high porosity and large surface area of PN-
51
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53 ceria significantly improved its catalytic activity (PN-ceria > HRP > non-porous ceria
54
55
56 nanorods > ceria octahedrons > ceria cubes > ceria nanoparticles). Furthermore, the
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peroxidase-like activity of PN-ceria was saturated with concentrations as low as
4
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6 0.3 μg/mL, which is a concentration significantly lower than the values reported for
7
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9 nanoceria (6 μg/mL)121 and Fe3O4 MNPs (40 μg/mL).104 Of note, the PN-ceria retained its
10
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12
catalytic activity after exposure to a temperature range from 30 to 200 °C or pH from 0
13
14 to 14, in contrast to natural HRP (Figure 10). The high stability of ceria towards harsh
15
16
17 conditions and their chemical inertness towards common acids and bases make them a
18
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20 highly attractive substitute for HRP in an ELISA system.
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35 Figure 10. Comparison of the stability of PN-ceria and HRP. a) PN-ceria and HRP were
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38 exposed to temperatures between 30 and 200 °C for 7 days and the peroxidase activity
39
40
41 was measured under standard conditions. b) Peroxidase activity measured under
42
43 standard conditions after 6 h for PN-ceria and HRP when exposed to different pH (0-14)
44
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46 solutions. Reproduced with permission ref 120. Copyright 2015 Elsevier.
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53 Since the oxidation of TMB is optimal in acidic conditions, immunoassays based on
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56 colorimetric responses from the oxidation of TMB limit the use of pH-labile
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biomolecules.122 To overcome this limitation, Asati et al. developed ceria-ELISA, where
4
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6 substrate conversion to yield quantifiable products could be performed at neutral pH.115
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9 Specifically, ampliflu (substrate) was used, which was oxidized by nanoceria at pH 7.0
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to generate the fluorescent product resorufin. In this case, no H2O2 is needed to oxidize
13
14 the fluorogenic substrates, offering an alternative to the H2O2-dependent nanoceria
15
16
17 activity. The ceria-ELISA with amplifu was successfully applied to detect the expression
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20 of folate receptor on lung carcinoma A549 cells and EpCAM on breast adenocarcinoma
21
22 MCF-7 cells. Detection of EpCAM from the colorimetric substrate TMB at pH 7.0 was
23
24
25 not possible as TMB is a poor substrate at this pH. When performed at pH 4.0,
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28 colorimetric ceria-ELISA using TMB identified EpCAM expression with a LOD of 3,600
29
30
cells. In contrast, fluorescent ceria-ELISA using ampliflu could detect EpCAM
31
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33 expression to as low as 300 cells.
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Platinum nanoparticles (Pt NPs)
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44 Pt NPs are another class of noble metal nanoparticle with peroxidase-like activity.
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47 Gao et al. developed a sandwich Pt-ELISA for the detection of human prostate-specific
48
49
50 antigen (PSA) in clinical samples, with an ultralow LOD of 0.8 pg/mL.123 Anti-PSA
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52 detection antibody was attached to Pt nanocubes via the covalent conjugation method.
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55 The colorimetric response was monitored based on the catalytic oxidation of TMB by Pt
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nanocubes. More recently, Loynachan et al. developed a paper-based porous platinum
4
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6 nanocatalysts (PtNCs) lateral flow immunoassay (LFIA) to detect p24,124 a key
7
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9 biomarker for HIV post infection. Although commercial immunoassay tests are
10
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available and have high sensitivity, the current LOD is still in the range of 10-15 pg/mL,
13
14 which is far above clinically relevant ranges of p24 during acute infection. Using
15
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17 catalytic amplification by PtNCs, the new assay enabled naked-eye detection of p24 in
18
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20 clinical samples with a sensitivity surpassing current commercial diagnostic kits for p24
21
22 (i.e. 0.8 pg/mL) under 20 min. In their work, PtNCs were conjugated with anti-HIV-1/2
23
24
25 antibody via the physical adsorption method. Then, antibody functionalized PtNCs and
26
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28 biotinylated nanobody fragments were mixed with serum samples. In the presence of
29
30
target analyte p24, PtNCs formed a complex with the target and biotinylated nanobody,
31
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33 which led to the deposition of PtNCs at the streptavidin test line. PtNCs catalyzed the
34
35
36 oxidation of 4-chloro-1-naphthol/3,3’-diaminobenzidine tetrahydrochloride (CN/DAB)
37
38
39
substrates in the presence of H2O2 to produce black precipitates at the test line (Figure
40
41 11a). Images of the lateral flow strips were acquired using a mobile phone and then
42
43
44 analyzed with ImageJ software. Through PtNC catalytic activity, an ultra-low
45
46
47 concentration from 1 to 10000 pg/mL of p24 could be detected (Figure 11b). This is a two
48
49 orders of magnitude broader dynamic range compared with the traditional ELISA and
50
51
52 fourth generation LFIA (Figure 11c).
53
54
55
56
57
58
59
36
1
2
3
4
5
6
7
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20
21
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23
24
25
26
27
28
29 Figure 11. a) Schematic illustration of a paper-based porous platinum nanocatalysts
30
31 (PtNCs) lateral flow immunoassay (LFIA) for the detection of HIV antigen (p24).
32
33
34 Antibody-labeled PtNCs complex with the target analyte and nanobody biotin, leading
35
36
37 to the deposition of PtNCs at the streptavidin test line. PtNCs catalyze the oxidation of
38
39
40
4-chloro-1-naphthol/3,3’-diaminobenzidine tetrahydrochloride (CN/DAB) substrates in
41
42 the presence of H2O2 to produce black precipitates at the test line. b) Linear dynamic
43
44
45 range for the detection of p24 across 4 orders of magnitude from 1 to 10000 pg/mL. c)
46
47
48 Schematic comparing the dynamic ranges of fourth generation LFIA, ELISA, and PtNC
49
50 LFIA. Reproduced with permission from ref 124. Copyright 2017 American Chemical
51
52
53 Society.
54
55
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37
1
2
3
4
5
6
7 Metal-organic frameworks (MOFs)
8
9
10
11 MOFs are an emerging class of porous material constructed by the chemical
12
13
14
coordination of metal ions and organic linkers,125-128 which have been reported to have
15
16 peroxidase-like activity.129-132 MOFs have large surface areas (typically ranging from
17
18
19 1000 to 10000 m2/g) and high porosity, a beneficial feature to enable an increased
20
21
22 interaction of substrates to the catalysts. Furthermore, their composition can be tuned to
23
24 tailor for specific functions or activities. In addition to peroxidase-like activity, studies
25
26
27 have also shown that encapsulation of natural enzymes within a MOF structure keeps
28
29
30 the enzymes functional under a wide range of (harsh) reaction conditions.133-134
31
32
33
Zhang et al. combined hemin and MOFs to form hemin-Au@MOF composites and
34
35
36 showed that peroxidase-like activity of hemin-Au@MOF (i.e. oxidation of TMB) was
37
38
39 four-fold higher compared with hemin-Au, hemin, and HRP. A MOF-based sandwich
40
41
42
ELISA system was developed and applied to detect alpha-fetoprotein (AFP), which is a
43
44 biomarker for liver cancer.135 In this study, hemin-Au@MOF labeled with anti-AFP
45
46
47 antibody bound to the analyte captured in the well plate and oxidized the substrate
48
49
50 TMB to produce a colorimetric response. To further enhance assay sensitivity, the
51
52 authors also applied a gold-catalyzed silver staining method. Through the dual-
53
54
55 catalyzation technique, as low as 0.02 ng/mL AFP in blood could be detected.
56
57
58
59
38
1
2
3
Wang et al. developed a sandwich ELISA for the detection of mouse IgG (mIgG)
4
5
6 based on Cu-MOF (Figure 12).136 Instead of conjugating antibodies onto the surface of
7
8
9 pre-formed MOFs, anti-IgG-Cu-MOF was derived via one-pot synthesis using anti-IgG,
10
11
12
CuCl2, and 4,4’-bipyridine. The loading efficiency of anti-IgG was determined to be
13
14 54%. Similar to MNPs and HRP, Cu-MOF activity is highly dependent on pH and H2O2
15
16
17 concentration. In this study, the optimal conditions for oxidation of TMB were found to
18
19
20 be pH 4.0 with 200 mM H2O2. This system was able to detect mIgG in serum at a linear
21
22 range of 1 to 100 ng/mL with a LOD of 0.34 ng/mL (3-fold higher sensitivity than HRP-
23
24
25 based ELISA). When the anti-IgG-Cu-MOFs were exposed to a high temperature of 80
26
27
28 °C, the peroxidase-like activity was retained. When subjected to trypsin, more than 80%
29
30
of the catalytic activity was still observed. This shows that the Cu-MOF host serves as
31
32
33 an effective shield for antibody against chemical and biological degradation. However,
34
35
36 the large surface area of MOFs could lead to non-specific binding and the authors
37
38
39
suggested that assay time longer than 40 min should be avoided.
40
41
42
43
44
45
46
47
48
49
50
51
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56
57
58
59
39
1
2
3
Figure 12. Schematic illustration of a sandwich ELISA for the detection of mouse IgG
4
5
6 (mIgG) using the peroxidase-like activity of Cu-MOF. Reproduced with permission
7
8
9 from ref 136. Copyright 2018 American Chemical Society.
10
11
12
13
14
15
16 Other metal and metal oxide nanoparticles such as palladium NPs (Pd NPs) and
17
18
19 manganese dioxide NPs (MnO2 NPs) have also been utilized as nanozymes in ELISA.
20
21
22 Xia et al. developed Pd-Ir core-shell nanocubes that exhibited high peroxidase mimetic
23
24 activity (3-fold higher than HRP) and stability.137 Using a sandwich ELISA method, Pd-
25
26
27 Ir nanocube achieved PSA detection with a LOD of 0.67 pg/mL, 110-fold more sensitive
28
29
30 than traditional ELISA.137 Xie and co-workers combined magnetic separation and
31
32
enrichment with MnO2 NPs to detect alpha-fetoprotein (AFP) from 1.56 to 1600 ng/mL
33
34
35 with a LOD of 21.6 pg/mL.138 In contrast, traditional ELISA based on natural enzymes
36
37
38 could only detect AFP in a linear range between 12.5 to 400 ng/mL with a LOD at 109
39
40
41
pg/mL.
42
43
44
45
46
47
48 Nanoparticles as signal transducers
49
50
51
52 Colorimetric analysis is the most common method used to measure the endpoint of
53
54
55 ELISA because it is simple and can easily be observed by the naked eye. However, it
56
57
58
59
40
1
2
3
may not be applicable for colored samples, such as blood, as it could interfere with the
4
5
6 colorimetric output. Therefore, variations of the standard ELISA are developed,
7
8
9 including fluorescence139-140 and chemiluminescence141-142 ELISA. They offer the
10
11
12
possibility of improving the sensitivity of the detection by 2−3 orders of magnitude
13
14 compared to the conventional colorimetric detection.
15
16
17
18
19
20
21
22 Quantum dots (QDs)
23
24
25 QDs are fluorescent inorganic semiconductor nanoparticles with diameters in the
26
27
28 range of 2-10 nm. They possess good photostability, high quantum yield, size-tunable
29
30
31 light emission, and are resistance against photobleaching, making them an attractive
32
33
alternative to fluorescent organic dyes in sensing applications.143-145 As opposed to
34
35
36 using an enzyme label, fluorescent QDs are conjugated to detection antibodies to form a
37
38
39 QD-ELISA system. A number of QD-ELISA has been reported, in which QD-antibody
40
41
42
conjugation was achieved via streptavidin-biotin interaction,146 EDC chemistry,147 trans-
43
44 cyclooctene-tetrazine chemistry,148 or using a hetero-bifunctional linker (e.g. sulfo-
45
46
47 SMCC).149 Herrera et al. developed a sandwich QD-ELISA for the detection of soluble
48
49
50 cytokine tumor necrosis factor-α (TNF-α) for concentrations as low as 60 attomolar (1
51
52 fg/mL).148 This is orders of magnitude more sensitive than the standard enzyme-based
53
54
55 ELISA and represents the most sensitive detection limit to date using a non-enzymatic
56
57
58
59
41
1
2
3
method. This sensitivity could be achieved because QDs exhibit superior photocatalytic
4
5
6 activity and emits bright fluorescence signals under visible-light irradiation owing to
7
8
9 their efficient light absorption and electron transfer. In their work, amine-terminated
10
11
12
QDs were first modified with NHS-tetrazine. Then, the QD-tetrazine were reacted with
13
14 trans-cyclooctene-modified detection antibody to form QD immunoconjugates. This
15
16
17 cycloaddition is fast, chemoseletive, adaptable to many metal nanoparticles, and does
18
19
20 not require a catalyst.150 The detection of TNF-α was performed in room temperature
21
22 for 30 min and the signal was obtained by monitoring QD fluorescence intensity at λex =
23
24
25 460 nm and λem = 590 nm. Other examples of QD-ELISA where QDs were conjugated to
26
27
28 detection antibodies include assays for CRP,151 imidaclothiz (insecticide),152 and
29
30
carcinoembryonic antigen.146
31
32
33
34 QDs have broad excitation spectra, allowing for simultaneous excitation of different
35
36
37 particle sizes at a single wavelength with emissions at multiple wavelengths. This
38
39
40
unique property offers an opportunity to develop a quantitative multianalyte assay in a
41
42 single reaction, as demonstrated by Peng et al.. Their contribution involved a
43
44
45 multiplexed QD-ELISA for the simultaneous detection of five compounds:
46
47
48 dexamethasone (DEX), medroxyprogesterone acetate (MPA), gentamicin (GM),
49
50 ceftiofur (CEF), and clonazepam (CZP).153 In this study, five corresponding antibodies
51
52
53 were coupled with QDs of different sizes with maximum emissions at 520 nm, 545 nm,
54
55
56 570 nm, 590 nm, and 635 nm via an avidin-biotin interaction (Figure 13a). Nanoparticle
57
58
59
42
1
2
3
conjugation onto antibody inevitably results in considerable loss of their functional
4
5
6 activity and binding capacity.154 As such, to achieve a high assay sensitivity, the authors
7
8
9 compared different QD-antibody conjugation techniques and found that avidin-biotin
10
11
12
interaction was superior to EDC/NHS crosslinking reaction, with 45-65% activity of the
13
14 antibody retained post conjugation. Using a single excitation at 335 nm, five standard
15
16
17 curves could be simultaneously obtained, and the established competition
18
19
20 fluoroimmunoassay QD-ELISA was used to analyze animal tissue matrices (pork
21
22 muscle). Another example was reported by Taranova et al., who developed a multicolor
23
24
25 QD-ELISA to detect and identify antibiotics (ofloxacin, chloramphenicol, and
26
27
28 streptomycin) in milk samples.155 Each antibody was labeled with QDs with maximum
29
30
emissions at 525, 585, or 625 nm, and the system was designed in a ‘traffic light’ format.
31
32
33 By this way, an analyte of interest could be identified based on the visible color of the
34
35
36 line. Moreover, the amount of the analytes present could be monitored based on the
37
38
39
fluorescence intensity of the lines (Figure 13b). The system exhibited high sensitivity,
40
41 with LODs for ofloxacin, chloramphenicol, and streptomycin determined to be 0.3, 0.1,
42
43
44 and 0.2 ng/mL, respectively. These values are 200 times lower than those achieved by
45
46
47 conventional enzyme-based ELISA using the same antibodies.
48
49
50
51
52
53
54
55
56
57
58
59
43
1
2
3
4
5
6
7
8
9
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21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40 Figure 13. a) Schematic illustration of a multiplexed QD-ELISA for the simultaneous
41
42
43 detection of five compounds: dexamethasone (DEX), medroxyprogesterone acetate
44
45
46
(MPA), gentamicin (GM), ceftiofur (CEF), and clonazepam (CZP). Five corresponding
47
48 antibodies are coupled with QDs of different sizes with maximum emissions at 520 nm,
49
50
51 545 nm, 570 nm, 590 nm, and 635 nm via avidin-biotin interaction. Reproduced with
52
53
54 permission from ref 153. Copyright 2009 Elsevier. b) Principle of a ‘traffic light’ QD-
55
56
57
58
59
44
1
2
3
ELISA for the simultaneous detection of three antibiotics: ofloxacin (OFL),
4
5
6 chloramphenicol (CAP), and streptomycin (STM). 1 – test zone for STM; 2 – test zone for
7
8
9 CAP; 3 – test zone for OFL; 4 – QD-antibody/STM; 5 – QD-antibody/CAP; 6 – QD-
10
11
12
antibody/OFL; 7 – control line. Each antibody is labeled with QDs with maximum
13
14 emissions at 525, 585, or 625 nm. (A) Test strip before the assay, (B) assay results for
15
16
17 sample containing STM, and (C) assay results for sample containing CAP and OFL.
18
19
20 Reproduced with permission from ref 155. Copyright 2014 Elsevier.
21
22
23
24
25
26
27 Instead of directly introducing QDs onto detection antibodies in an ELISA, Pavlov
28
29
30 and co-workers developed an immunoassay for the detection of superoxide dismutase 2
31
32
(SOD2) with signal output based on in situ generation of fluorescent QDs.156 Increased
33
34
35 SOD2 expression is a biomarker for tumor progression and metastasis in prostate,
36
37
38 colon, and lung cancer.157-158 Anti-SOD2 antibody was functionalized onto a polyvinyl
39
40
41
chloride microbead via streptavidin-biotin complexes. BSA was used to block non-
42
43 specific adsorption. In the presence of SOD2 and ALP-conjugated anti-SOD2 antibody,
44
45
46 the complexes formed. The immobilized ALP hydrolyzed p-nitrophenyl phosphate to
47
48
49 p-nitrophenol and orthophosphate ions, leading to the rapid formation of phosphate-
50
51 stabilized CdS QDs on the surface of microbeads (Figure 14a). The QDs served as a
52
53
54 signal transducer which emitted fluorescence under excitation with 320 nm light. Based
55
56
57
58
59
45
1
2
3
on the fluorogenic analysis, SOD2 could be detected in a linear range between 0 to 11
4
5
6 ng/mL, with LOD of 0.52 ng/mL (Figure 14b). Compared to the standard chromogenic
7
8
9 analysis, the fluorescence method has 2.5 times better detection limit.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50 Figure 14. a) Schematic illustration of a QD-ELISA for the detection of superoxide
51
52
53 dismutase 2 (SOD2) based on in situ generation of fluorescent QDs. (A) Anti-SOD2
54
55 antibody conjugation onto microbeads; (B) immobilization of alkaline phosphatase
56
57
58
59
46
1
2
3
(ALP)-conjugated anti-SOD2 antibody mediated by SOD2; (C) formation of phosphate-
4
5
6 stabilized CdS QDs on the surface of microbeads. b) (A) Fluorescence emission spectra
7
8
9 of CdS QDs in various concentrations of SOD2; (B) Calibration curve of SOD2.
10
11
12
Reproduced with permission from ref 156. Copyright 2016 American Chemical Society.
13
14
15
16
17
18
19 Upconversion nanoparticles (UCNPs)
20
21
22
23 UCNPs are a new generation of nanoscale particles which display unique anti-
24
25 Stokes optical properties. By varying the lanthanide doping, UCNPs can exhibit
26
27
28 wavelength-selective upconversion, such as near-infrared (NIR) to shorter NIR, visible
29
30
31 (including blue, green, red), or UV. For this reason, UCNPs can achieve minimum
32
33
autofluorescence and background noise, avoid photobleaching and photoblinking, and
34
35
36 display deep and non-invasive tissue penetration. This makes UCNPs an excellent
37
38
39 alternative compared with conventional fluorophores, such as organic dyes, metal
40
41
42
complexes, or QDs.159 Moreover, UCNPs can be easily tailored in regards to their
43
44 morphology (cubic, spherical, or rod-shaped), size (1-100nm), emission (shorter NIR to
45
46
47 the visible range) and composition.160-161 With these distinct photophysical properties,
48
49
50 UCNPs can serve in a broad range of nanosensing platforms. Considering most of the
51
52 lab-prepared UCNPs are originally hydrophobic, they are not suitable for bioanalytical
53
54
55 applications. Therefore, surface modification is necessary to introduce a hydrophilic
56
57
58
59
47
1
2
3
surface with recognition moieties. Multiple strategies are available to accomplish this
4
5
6 including ligand exchange, ligand oxidation, ligand interaction, silica shell deposition,
7
8
9 and bioconjugation.162
10
11
12
13
Hlaváček et al. developed a competitive upconversion-linked immunosorbent
14
15 assay (ULISA) to detect a nonsteroidal anti-inflammatory drug, diclofenac (DCF).163 In
16
17
18 this study, carboxylated silica-coated UCNPs were conjugated with anti-mouse IgG
19
20
21 antibodies via EDC/NHS reactions. It was determined that there were 33 anti-IgG
22
23 molecules per 42.5 nm-diameter UCNP. The attachment of anti-mouse IgG-UCNP
24
25
26 secondary antibody conjugate to anti-DCF antibody in ULISA was monitored by 980
27
28
29 nm laser excitation (Figure 15a). In this case, the luminescent signal was inversely
30
31
proportional to the DCF concentration. Compared with traditional ELISA, the reported
32
33
34 ULISA achieved five times higher LOD (0.05 ng/mL vs 0.01 ng/mL CDF, Figure 15b).
35
36
37 The same research group also developed an ultrasensitive sandwich ULISA for
38
39
40
honeybee pathogen Melissococcus plutonius with UCNPs as the luminescent reporter.164
41
42 This system led to a LOD of 340 CFU/mL, which is 400 times lower than conventional
43
44
45 ELISA.
46
47
48
49
50
51
52
53
54
55
56
57
58
59
48
1
2
3
Figure 15. a) Schematic illustration of direct competitive ULISA for the detection of
4
5
6 diclofenac (DCF). A well plate is first coated with bovine serum albumin-DCF (BSA-
7
8
9 DCF). Then different concentration series of DCF and anti-DCF antibody are added to
10
11
12
the wells. The attachment of anti-mouse IgG-UCNP secondary antibody conjugate to
13
14 anti-DCF antibody in ULISA is monitored by 980 nm laser excitation. b) Calibration
15
16
17 curves of ULISA for DCF (red) compared with the traditional ELISA (black).
18
19
20 Reproduced with permission ref 163. Copyright 2016 American Chemical Society.
21
22
23
24
25
26
27 In principle, UCNPs are utilized in an immunoassay as a luminescence reporter to
28
29
30 replace the traditional enzyme-catalyzed oxidation of substrates to colorimetric
31
32
products. However, just like other classes of nanoparticles, UCNPs are susceptible to
33
34
35 non-specific binding, which generates high background signal and therefore may
36
37
38 reduce the signal-to-noise ratio. To date, optimizing the surface chemistry of UCNPs or
39
40
41
adding blocking proteins (e.g. BSA) are considered a useful method to solve this issue.
42
43 Polymers can also be used as a blocking agent in an immunoassay, as shown by
44
45
46 Lahtinen and co-workers.165 The authors coated UCNPs with poly(acrylic acid) (PAA),
47
48
49 which served two functions: i) carboxylic groups of PAA enabled antibody
50
51 functionalization via covalent conjugation method and ii) free PAA was added in assay
52
53
54 buffer to prevent UCNP non-specific binding. In this study, a sandwich ULISA was
55
56
57
58
59
49
1
2
3
developed for the detection of disease biomarkers, cardiac troponin I (cTnI) and thyroid
4
5
6 stimulating hormone (TSH). The luminescence signal was detected at 540 nm under 980
7
8
9 nm laser excitation. The results showed that higher sensitivity was achieved by adding
10
11
12
free PAA in the buffer. The limits of detection of cTnI and TSH decreased from 2.1 ng/L
13
14 to 0.48 ng/L, and from 0.07 mIU/L to 0.02 mIU/L, respectively.
15
16
17
18 It is worth noting that QDs and UCNPs possess advantages over traditional
19
20
21 fluorophores. QDs are significantly brighter and UCNPs are resistant to interference.
22
23 However, the drawbacks are that QDs (especially CdSe-based QDs) are relatively toxic
24
25
26 and UCNPs require specialized excitation sources (880 nm or 980 nm), which hinder
27
28
29 their popularization in immunoassays. Therefore, in addition to these two signal
30
31
transducers, carbon quantum dots and graphene quantum dots can be employed as the
32
33
34 alternatives because they have several advantages such as lower toxicity, excellent
35
36
37 water solubility, and ease of synthesis.166-168
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
50
1
2
3 Table 1 Metal and metal oxide nanoparticle-enhanced ELISA discussed in this review
4
5 Comparison with
6 Types of
Types of Enzyme traditional Re
7 nanoparticle Signal Analyte Detection range LOD
8 ELISA used ELISA f
s
9 (LOD
10 improvement)
11 Nanoparticles as carriers
12
13
14 Colorimetri
15 AuNPs Sandwich CA15-3 HRP 0 – 60 U/mL 0.012 2-fold 43
c
16 OD·mL/U
17
18 Colorimetri
AuNPs Sandwich Gliadin HRP 102 – 106 pg/mL 180 pg/mL 7-fold 47
19
c
20
21
22 Colorimetri
AuNPs Sandwich CRP HRP 0.1 – 1000 ng/mL 0.1 ng/mL 100-fold 61
23 c
24
25 Respirator
26 Colorimetri 62
AuNPs Sandwich y syncytial HRP 0.5 – 50 pg/mL 0.5 pg/mL 50-fold
27 c
28 virus
29
30 Colorimetri Procalcito 63
31 AuNPs Sandwich HRP 0.02 – 20 ng/mL 20 pg/mL 5-fold
c nin
32
33
Indirect
34 Colorimetri 64
35 AuNPs competitiv Fumonisin HRP 0.01 – 1000 μg/L 78 ng/L 10-fold
c
36 e
37
38 AuNP Colorimetri
39 Direct EGFR HRP – 5000 cells 13-fold 65
nanoshells c
40
41
42
43
44
46 51
47
1
2
3
AuNP-
4 Colorimetri
5 PAMAM Sandwich hCG HRP 0.1 – 6.4 IU/L 0.03 IU/L 27-fold 66
c
6 dendrimers
7
8 Colorimetri Antithrom
AuNPs Indirect ALP 125-fold 67
9 0.0768 – 9.6 ng 76.8 pg
c bin
10
11
12 Colorimetri Aflatoxin
MNPs Sandwich HRP 0.002 – 0.2 ng/mL 2 pg/mL 10-fold 73
13 c B1
14
15 MNP- Ara h3/4
16 Colorimetri
PAMAM Sandwich peanut HRP 2.5 – 15 mg/kg 0.2 mg/kg 3-fold 44
17 c
18 dendrimers allergens
19
20 Fluorescenc IgM- 0.04
21 MNP-PDA Sandwich ALP 0.03 – 1 μg/mL 700-fold 80
e dengue μg/mL
22
23
Colorimetri 3.85 fg/mL – 7.7
24 SNPs Sandwich Insulin HRP 3.85 fg/mL 2000-fold 81
25 c pg/mL
26
27 Mesoporous Colorimetri
28 Sandwich hIgG GO 0.1 – 1000 ng/mL 0.5 ng/mL 20-fold 86
SNPs c
29
30
Nanoparticles as enzyme mimics
31
32
33 Comparison with
34 Types of traditional
35 Types of Re
nanoparticle Signal Analyte Detection range LOD ELISA
36 ELISA f
s (LOD
37
38 improvement)
39
40
41
42
43
44
46 52
47
1
2
3
Colorimetri
4 AuNPs Sandwich hIgG 0.7 – 100 ng/mL 0.3 ng/mL 5-fold 98
5 c
6
7 Graphene- Colorimetri 92.7
8
Direct Norovirus 100 pg/mL – 10 μg/mL 112-fold 103
AuNPs c pg/mL
9
10
ZnFe2O4@M Colorimetri
11 Sandwich CEA 0.005 – 30 ng/mL 77-fold* 110
12 WNT c 2.6 pg/mL

13
14 Colorimetri 0.01
15 Ceria Sandwich CA15-3 0.01 ng/mL – 100 μg/mL 10-fold 120
c ng/mL
16
17
Fluorescenc
18 Ceria Direct EpCAM 300 – 6000 cells 300 cells N/A 115
19 e
20
21 Colorimetri
Pt nanocubes Sandwich PSA 20 – 2000 pg/mL 10-fold 123
22 c 0.8 pg/mL
23
24
25
Colorimetri 0.8
Pt NPs Sandwich HIV p24 1 – 10000 pg/mL 1.3-fold* 124
26 c pg/mL
27
28 Hemin- Colorimetri 0.020
29 Sandwich AFP 0.080 – 43 ng/mL 5-fold 135
Au@MOF c ng/mL
30
31
Colorimetri
32 Cu-MOF Sandwich mIgG 1 – 100 ng/mL 0.34 3-fold 136
33 c
34
ng/mL
35 Colorimetri
36 Pd NPs Sandwich PSA 2 – 1200 pg/mL 0.67 110-fold 137
c
37 pg/mL
38
Colorimetri
39 MnO2 NPs Sandwich Alpha- 1.56 – 1600 ng/mL 21.6 5-fold 138
40 c
41
fetoprotei pg/mL
42
43
44
46 53
47
1
2
3
4
n
5
6 Nanoparticles as signal transducers (fluorescence or luminescence)
7
8 Comparison with
9 Types of traditional
10 Types of Re
nanoparticle Signal Analyte Detection range LOD ELISA
11 ELISA f
12 s (LOD
13 improvement)
14
15 Fluorescenc
16 QDs Sandwich TNF-α 0.001 – 1000 pg/mL 1 fg/mL 20-fold 148
e
17
18
19 OFL 1.5 – 200 ng/mL 0.3 ng/mL
20 QDs Competiti Fluorescenc
21 CAP 0.14 – 10 ng/mL 0.12 ng/mL 80- to 200-fold 155
ve e
22
23 STM 0.3 – 500 ng/mL 0.2 ng/mL
24
25 Fluorescenc 156
26
QDs Sandwich SOD2 0 – 11 ng/mL 2.5-fold
e 0.52 ng/mL
27
28
Luminescen 163
29 UCNPs Sandwich DCF 0.05 – 10 ng/mL 0.05 ng/mL 5-fold
30 ce
31
32 Melissococc
Luminescen 164
33 UCNPs Sandwich us 340 – 109 CFU/mL 340 400-fold
34 ce
plutonius CFU/mL
35
36
37 *Compared with commercialized ELISA kit
38
39
40
41
42
43
44
46 54
47
1
2
3
4 Conclusions
5
6
7
8 In this review, we highlight recent successes of metal and metal oxide nanoparticles
9
10 in improving the performance of ELISA, in particular towards improving assay
11
12
13 sensitivity and stability as well as shortening assay time. Nanoparticles have large
14
15
16 surface area-to-volume ratio that allow the attachment of multiple enzyme-conjugated
17
18
antibodies (e.g. up to 20 HRP per nanoparticle), which generates an amplified
19
20
21 colorimetric signal compared with a single enzyme-conjugated antibody. To date,
22
23
24 nanoparticle-modified ELISA has led to an ultrasensitive assay with three orders of
25
26
27
magnitude enhancement of sensitivity compared with traditional ELISA and LOD
28
29 down to fg/mL in human serum. Meanwhile, nanoparticles can serve as a robust
30
31
32 platform to increase the stability of natural enzymes and antibodies. Antibody-
33
34
35 nanoparticle conjugates still retained their initial activity even after 1-month storage at
36
37 room temperature.
38
39
40
41 A variety of conjugation chemistries have been reported to functionalize
42
43
44
nanoparticles with antibodies and enzymes. There is no one-size-fits-all approach and it
45
46 is important to note that functionalization methods influence antibody activity and
47
48
49 therefore antigen-sensing ability. Critical to the optimization of antibody-nanoparticle
50
51
52 functionalization is the antibody loading, orientation of the antibody, minimum
53
54 antibody modification, and accessibility of antigen-binding sites. Uniformly oriented
55
56
57
58
59
55
1
2
3
antibodies will provide high assay sensitivity and consistency for the construction of
4
5
6 nanomaterials-based ELISA. Therefore, antibody loading and accessibility should be
7
8
9 evaluated, for example using a radio-labeled assay,169 a fluorescence-based method,170
10
11
12
or an enzyme-based analytical method,60 which can be easily implemented as a
13
14 standardized approach when reporting antibody-nanoparticle conjugation.
15
16
17
18 Due to the simple preparation, low cost, and excellent stability against high
19
20
21
temperatures, extreme pH conditions, and biological degradation, nanozymes could
22
23 gradually replace the use of natural enzymes in ELISA. With nanozymes, it is even
24
25
26 possible to oxidize substrates to yield colorimetric products without relying on unstable
27
28
29 H2O2. In 2018, two clinical nanozyme-based immunoassays, blood transferrin test and
30
31 fecal occult-blood test, were approved by the China Food and Drug Administration.171
32
33
34 To date, nanozymes are broadly divided into two categories: oxidoreductrase family
35
36
37 (e.g. peroxidase, oxidase, catalase, superoxide dismutase) and hydrolase family (e.g.
38
39 nuclease, protease, esterase, phosphatase). Many studies report nanozyme-ELISA by
40
41
42 applying the peroxidase-like activity of the nanoparticles. In addition to HRP, two other
43
44
45 common enzymes used in colorimetric immunoassays are ALP and β-galactosidase.
46
47
Nanoparticles that mimic the function of these enzymes will broaden the avenue of
48
49
50 nanozyme-ELISA. Although the progress made to date is encouraging with remarkable
51
52
53 improved assay sensitivity, there are still challenges to overcome. Unlike natural
54
55
56 enzymes, nanozymes do not catalyze one specific substrate, i.e. they have multi enzyme
57
58
59
56
1
2
3
mimetic activity.171 Depending on the composition of the samples, the poor substrate
4
5
6 selectivity may potentially lead to false measurements in assays. Understanding the
7
8
9 catalytic mechanisms of nanozymes (e.g. using density functional theory (DFT)
10
11
12
calculations172-173) will further improve both assay sensitivity and substrate selectivity.
13
14 Although nanozymes can be mass-produced easily, improving batch-to-batch
15
16
17 reproducibility is vital. While ultralow detection down to femto level has been
18
19
20 achieved, small changes in analyte concentration can result in undistinguishable
21
22 colorimetric or fluorescence signal. Therefore, detection could be coupled with
23
24
25 smartphone technologies,174-175 a portable benchtop reader,176 or a high-resolution
26
27
28 detector177 for better signal readout and, possibly, to further lower LOD.
29
30
31 We believe the advancement of nanomaterials-based ELISA could revolutionize
32
33
34 analyte detection and screening in an affordable and easily accessible way. We hope
35
36
37 that this review stimulates further research activity in this field.
38
39
40
41
42 AUTHOR INFORMATION
43
44
45 Corresponding Author
46
47
48 *E-mail: rona.chandrawati@unsw.edu.au
49
50
51
52 Notes
53
54
55 The authors declare no competing financial interest.
56
57
58
59
57
1
2
3
4
5
6 ACKNOWLEDGMENTS
7
8
9 R.C. acknowledges the support from the Australian Research Council Discovery Early
10
11
12
Career Researcher Award (ARC DECRA DE170100068) and the UNSW Scientia
13
14 Fellowship. We thank Federico Mazur for critical reading of the manuscript.
15
16
17
18
19
20
21
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Metal and Metal Oxide Nanoparticles To Enhance The Performance of Enzyme-Linked Immunosorbent Assay (ELISA) (ACS Applied Nano Materials) (2019)

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