I.

Materials
An Alexa Fluor 488 protein labeling kit was purchased from Invitrogen
(Carlsbad, CA).
GOx (E.C. 1.1.3.4) type X-S from Aspergillus niger (G7141, optimal pH
5.0−7.0, and optimum temperature 40−60 °C).
Horseradish peroxidase (HRP) type VI from horseradish roots, sodium
chloride (NaCl),
sodium bicarbonate (NaHCO3),
potassium cyanide (KCN),
potassium chloride (KCl),
phosphate-buffered saline (PBS) tablets (10 mM, pH 7.2), sulfuric acid
(H2SO4),
copper sulfate (CuSO4),
gold chloride trihydrate (HAuCl4·3H2O),
ferrocene-methanol (FcMeOH),
o-phenylenediamine (OPD),
Whatman Anotop 25 syringe filters with a 20 nm filtering size were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
AuNP colloidal solution (20 nm) was purchased from BBI Solutions
(Cardiff, U.K.).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-
glycero-3 phosphoethanolamine (DOPE), and cholesterol were purchased
from Avanti Polar Lipids Inc., U.S.A.
Milli-Q water with resistivity ≥18 MΩ·cm cm was used in all experiments.
II. Theoretical Calculations Estimate the Number of GOx Needed to
Full Cover the AuNP Surface.
To characterize the GOx in terms of macromolecular shape changes after
adsorbing it tovthe surface of AuNPs, theoretical calculations of the
estimated maximum number of GOx that can fit at the surface of a 20 nm
AuNP while keeping the original dimension of the enzyme were performed
using published data on GOx size and calculated for the different
geometrical ways enzymes can pack onto the AuNP surface
 Figure 1: Estimation of the number of enzymes that can fit and fully cover the surface of an
AuNP. (A) Illustration of cases when considering the enzyme shape as a sphere; an elliptical
shaped macromolecule attaching by its short-side and an elliptical enzyme attaching by its long-
side. (B) Mathematic calculation of the number of GOx that can fully cover the surface of a 20
nm AuNP at different dimension sizes. Schematics are not drawn to scale.

Where a is the semimajor axis, and b is the semiminor axis of the enzyme.
Considering the available surface area of the AuNP, AAuNP, for GOx to
bind, the maximum number of GOx that can theoretically fit and fully cover
the available surface area of a 20 nm AuNP depending on the average shape
and direction for the enzyme to bind to the AuNP surface was then
estimated.
A AuNP
Number of GOx per AuNP = A
E

III. Preparation of GOx/AuNP Conjugates.

The process described in this passage involves the preparation of

GOx/AuNP (Glucose Oxidase/Gold Nanoparticle) conjugates. Below is the
experimental procedure for preparing the GOx/AuNP conjugate:

1. Preparation of GOx/AuNP Conjugates: The goal is to create a

conjugation between Glucose Oxidase (GOx) and Gold Nanoparticles
(AuNP). This is achieved by allowing GOx to self-adsorb onto the
surface of AuNPs.

2. Binding Interaction: The interaction between negatively charged GOx

and AuNP surfaces is achieved through various weak forces, including
van der Waals forces, hydrogen bonds, and hydrophobic interactions.
Additionally, covalent thiol bonds at one of the cysteine-rich sides of
GOx contribute to strong binding to gold.
 3. Characteristics of AuNPs: The AuNPs used have a size of 20 nm and
are suspended in water with citrate as a capping agent. Their size
variation is minimal, with a coefficient of variation less than or equal to
8%.

4. Preparation of Enzyme-to-AuNP Ratios: GOx/AuNP conjugates with

varying enzyme-to-AuNP molar ratios are created by adding different-
sized aliquots from a 2 mg/mL GOx stock solution in 10 mM NaHCO3
buffer, pH 8.2, to a fixed volume of 1 nM AuNP solution in 2 mL tubes.

5. Incubation: The resulting mixture is incubated in the dark at room

temperature for 1.5 hours.

6. Centrifugation and Isolation: After the incubation process, the

GOx/AuNP conjugates are isolated by centrifugation at 10,621g for 30
minutes at 4 °C. This separates the bound GOx/AuNP conjugates from
unbound GOx.

7. Supernatant Removal: The supernatant, which contains the unbound

GOx, is carefully removed and transferred to separate tubes for later
quantification of the enzyme.

8. Resuspension: A portion of the pelleted GOx/AuNP conjugates (50 μL)

is left in the bottom of the tube and is resuspended into 10 mM NaHCO3
buffer to replace the same volume as the extracted supernatant. This
resuspension process is repeated two times.

9. Cleaning and Elimination: Any AuNPs that remained uncoated or

partially coated and aggregated are eliminated from further analysis. They
tend to stick to the wall of the test tubes.

IV. Flocculation Assays.

To determine the ratio of enzyme-to-AuNP required in a solution to fully

cover the surface of AuNPs during the enzyme-AuNP conjugation process, a
flocculation assay was performed. In summary, the assay was designed as
follows:

1. Sample Preparation: The assay was designed by preparing samples

containing GOx/AuNP conjugates with various enzyme-to-AuNP ratios.

2. Adding NaCl: For each sample, a solution of NaCl was added to achieve
a final concentration of 0.2 M. This high-salt solution reduces the electric
double layer at the surface of AuNPs, causing colloidal destabilization.
 3. Colloid Precipitation: Consequently, if there is an insufficient amount of
enzyme to cover the AuNP surface, the AuNPs aggregate in the solution,
leading to colloidal precipitation.

4. Preventing Precipitation: However, if enough enzyme coats the AuNP

surface, it creates a physical barrier for the AuNPs, preventing them from
aggregating into precipitates.

5. Monitoring with UV-Vis Spectroscopy: This process can be monitored

using UV-Vis spectroscopy. Sufficiently protected GOx/AuNP
conjugates display a plasmonic peak that overlaps with the peak
wavelength of 524 nm for bare 20 nm AuNPs, which serves as a
reference sample. In contrast, aggregates formed from inadequately
protected AuNPs shift towards the red spectrum.

6. Absorbance Spectrum Measurement: Therefore, after incubating the

samples in a high-salt solution in the dark for 20 minutes, an absorbance
spectrum ranging from 400 to 800 nm was measured using a Cary 4000
and Cary 5000 UV-Vis spectrophotometer (Agilent Technologies, USA)
and a Hellma quartz Suprasil cuvette QS 10 mm as a UV-Vis sample
container.

7. Determining Enzyme Ratio: By monitoring the individual UV-Vis

spectra of the conjugate samples, the minimum amount of enzyme
required to fully cover the AuNP surface can be determined by
identifying spectra where there is a complete overlap of peaks with bare
AuNPs in Milli-Q water.

V. Enzyme Labeling.

For the fluorescent quantification assay, Glucose Oxidase (GOx) was

prelabeled with an Alexa Fluor 488 dye using an Alexa Fluor 488 protein
labeling kit from Invitrogen:

1. Preparation of GOx Solution: A solution of GOx with a concentration

of 2 mg/mL was prepared in 0.1 M NaHCO3 at pH 8.

2. Labeling with Alexa Fluor 488 Dye: The GOx solution was incubated
with the Alexa Fluor 488 dye for one hour in the dark while stirring. This
process allowed the dye to bind to the GOx molecules, resulting in
fluorescently labeled GOx.

3. Separation of Nonbound Dye: Any fluorescent tags that remained

unbound were separated from the labeled enzyme. This separation was
 achieved using a size exclusion separation column provided by the
labeling kit.

4. Determination of Protein Concentration: The concentration of the

labeled GOx protein and the degree of fluorophore labeling were
determined according to the protocol provided in the labeling kit.
Extinction coefficients, which are essential for quantifying the labeled
protein's fluorescence, were also determined through the kit's instructions.

VI. Indirect and Direct Quantification of Enzyme Bound to the

AuNP Surface.

The passage describes an experimental method used to determine the

number of enzymes attached to the surface of gold nanoparticles
(AuNPs). Two quantification methods, indirect and direct, were
employed simultaneously.

1. Indirect Quantification Method:

 Fluorescently labeled Glucose Oxidase (GOx) was used in the

conjugation process with AuNPs.
 After the conjugation, a centrifugation step was performed to separate
non-bound enzymes from the GOx/AuNP conjugates.
 The amount of non-bound enzymes in the supernatant solution was
quantified using a Cary Eclipse fluorescent spectrophotometer.
 Calculations were made to relate the amount of enzymes measured free in
solution to the amount added during the conjugation process, determining
the amount of enzymes bound to the AuNP surface.
 A new calibration curve for the labeled GOx was created for each batch
of labeled enzymes and for each assay.

2. Direct Quantification Method:

 The amount of GOx immobilized onto the AuNP surface was determined
using this method.
 After the centrifugation step, the GOx/AuNP conjugates were subjected
to potassium cyanide (KCN), which dissolved the AuNPs and released
the enzymes into solution.
 50 μL of freshly made 25 mM KCN was added to 200 μL of the
conjugate pellet solution, followed by a half-hour incubation in the dark.
 The sample solutions turned completely optically clear as the colloidal
particles dissolved.
 The free GOx in solution was then quantified using fluorimetry.
 To determine the average number of enzymes per AuNP, the amount of
immobilized GOx was correlated with the number of non-aggregated
AuNPs in each sample solution.

3. AuNP Concentration Measurement:

 The concentration of non-aggregated AuNPs in each sample was

determined using a spectroscopy-based method established by Haiss et
al., which involves measuring the absorbance at 450 nm using a UV-vis
spectrophotometer.
 The AuNP concentration was calculated using a formula that takes into
account the diameter of the AuNPs and the absorbance at 450 nm.

4. Calculation of Average Enzymes per AuNP:

 Finally, the average number of enzymes per AuNP in each sample was
calculated using both the indirect and direct quantification methods of
enzyme binding to AuNP surfaces.
 This information was related to the measured number of non-aggregated
AuNPs in solution, providing an average amount of GOx adsorbed per
AuNP in each sample.

VII. DLS Measurements of Conjugate Size.

This passage describes a procedure for monitoring the size of

conjugates formed during the process of conjugating enzymes with gold
nanoparticles (AuNPs) :
1. Objective: The goal is to monitor the size of conjugates formed when
varying the number of enzymes added during the AuNP conjugation
process.
2. Instrumentation: The hydrodynamic diameter (Dhyd) of AuNPs and
GOx/AuNP conjugates is measured using a Zetasizer Nano ZS, a device
made by Malvern Instruments Ltd. It uses a He−Ne laser (633 nm) as a
light source.
3. Cuvettes: Small volume (70 μL) cuvettes made by BRAND GMBH + CO
KG, Germany, are used for the measurements.
4. Data Acquisition: For each sample, three measurements are taken, and
each measurement consists of 12−15 runs, providing multiple data points
for accuracy.
 5. Measurement Angle: Backscatter detection is used with an angle of 173°
at room temperature.
6. Data Analysis: Zetasizer software version 7.10 is used to analyze the
data. The reported values are based on the Z-average size, which is an
intensity mean obtained via cumulative analysis.
7. Timing: The dynamic light scattering (DLS) measurements are conducted
6 hours after the GOx/AuNP conjugation process.
8. Buffer Solution: To ensure accurate measurements and eliminate
artifacts, the NaHCO3 buffer solution is filtered using a 20 nm syringe
filter before use.
VIII. NTA Measurements of the Conjugate Size.

The passage describes the process of measuring the size of colloidal

particles, specifically gold nanoparticles (AuNPs) and glucose oxidase/gold
nanoparticle (GOx/AuNP) conjugates, using a Malvern NanoSight LM10
microscope:

1. Instrumentation: The measurements were carried out using a Malvern

NanoSight LM10 microscope, which was equipped with a temperature-
controlled sample chamber. The microscope had a 488 nm (blue) laser
and a highly sensitive C11440-50B/A11893-02 sCMOS camera from
Hamamatsu Photonics K.K., Japan.

2. Measurement Process: The microscope was used to track the scattered

light produced by individual AuNPs and GOx/AuNP conjugates. This
scattered light was observed under Brownian motion.

3. Software: NTA 3.1 software was used for data analysis. The software
performed 5 runs of 60-second video recording to capture the motion of
colloidal particles, which were later analyzed to determine the average
particle size.

4. Conditions: All measurements were conducted at room temperature.

Measurements were taken immediately after a three-step centrifugation
cleaning process of the GOx/AuNP conjugates and also 6 hours after the
GOx/AuNP conjugation process.

5. Artifact Elimination: To ensure accurate measurements and eliminate

artifacts, the NaHCO3 buffer used in the process was filtered using 20 nm
syringe filters before application.
 IX. Enzyme Activity Assay.

The passage describes an experiment aimed at determining the

retained enzyme activity of glucose oxidase (GOx) after it is immobilized
onto the surface of gold nanoparticles (AuNP) compared to the enzymatic
activity of free GOx in a solution:

1. Assay Method: The researchers used a UV-vis spectrophotometer-based

assay to measure enzyme activity.

2. Catalytic Reactions: In the assay, glucose is catalyzed by GOx to

produce gluconic acid and hydrogen peroxide (H2O2). Subsequently, in
the presence of an additional enzyme, horseradish peroxidase (HRP),
H2O2 is converted to produce 2,3-diaminophenazine (DAP), which can be
quantified spectrophotometrically.

3. Measurement of DAP: DAP's absorbance is measured at 492 nm, using

the Lambert-Beers law and a molar absorptivity coefficient (ε 492 = 67,143
M−1 cm−1) for DAP at 429 nm.

4. Conjugation Process: Conjugates of GOx and AuNP were prepared with

a 100:1 molar ratio of GOx to AuNP.

5. Assay Setup: The assay setup involved adding specific volumes of GOx,
HRP, OPD (o-phenylenediamine), and glucose solutions to a 24-well
plate. The reaction was initiated by adding glucose and stopped after 20
seconds by adding HCl.

6. Optimization: Experimental conditions, including the molar ratio of

glucose to GOx and reaction times, were optimized to ensure a maximal
reaction rate and to avoid substrate inhibition.

7. Caution: The experiments were conducted in the dark at room

temperature within a short timeframe because HRP and OPD are sensitive
to light, and OPD can undergo autocatalysis.
8. Enzyme Stability: In addition to comparing the activities of free GOx
and immobilized GOx, the study also investigated the stability of the
enzyme. The enzyme activity was monitored from day 0 up to 14 days
after the conjugation process was initiated.

The enzyme activity for both immobilized and free GOx was calculated
using eq 5.

where nDAP is the amount of produced DAP in moles, t is the reaction time
in minutes, and mGOx is the mass of GOx added into the reaction.
X. Glucose Liposome Preparation.

This passage describes the preparation of glucose liposomes for a

scientific experiment. Here's a summary of the process:

1. Ingredients: The glucose liposomes were prepared using DOPC (1,2-

dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-
3-phosphoethanolamine), and cholesterol solutions in a specific molar
ratio of 39:21:40.

2. Lipid Film Formation: These lipid solutions were added to a round

flask, and the mixture was stirred under N2 gas for 3 hours. This stirring
process led to the formation of a thin lipid film on the internal wall of the
flask.

3. Rehydration: To create the glucose liposomes, the lipid film was

rehydrated by adding a 200 mM glucose solution dissolved in 10 mM
HEPES buffer (pH 7.4) to it. This rehydration process took place for 30
minutes at room temperature, resulting in a final concentration of
approximately 1.25 mg/mL for total lipids.

4. Freeze-Thaw Cycles: The liposome solution was subjected to five cycles

of freeze and thaw. This involved alternately placing the solution into
liquid nitrogen and water at room temperature. This process was
performed to completely trap glucose inside the liposomes.

5. Size Unification: The size of the liposomes was made uniform by

passing the liposome suspension through a polycarbonate membrane with
a pore size of 400 nm. This was done 21 times under a constant pressure
of 1 bar in N2 gas at room temperature, using an Avanti Mini-Extruder.
 6. Removal of Unloaded Glucose: Any glucose that was not encapsulated
within the liposomes was removed. This step was carried out using
illustra MicroSpin S-200 HR columns.

7. Osmotic Pressure Adjustment: The osmotic pressure of the dilution

solution, which would be used for later electrochemical measurements,
was adjusted to 236 mOsm/kg by dissolving NaCl in 10 mM HEPES
buffer (pH 7.4).

XI. Amperometric Measurements of the Ultrafast Glucose

Biosensor Using a Glucose Liposome.

The passage describes the fabrication of glucose biosensors and related tests:

1. Sensor Fabrication: Glucose biosensors were created by inserting

individual 33 μm diameter carbon fibers into single borosilicate glass
capillaries with 1.2 mm outer diameter and 0.69 mm inner diameter
(Sutter Instrument Co., Novato, CA, USA). A micropipette puller (model
P-1000, Sutter Instrument Co., Novato, USA) was then used to pull the
carbon fiber-filled glass capillary from the middle into two electrodes
with fine tapered tips. All electrode tips were immersed in epoxy
solutions (Epo-Tek 301, Epoxy Technology, Billerica, MA, USA). Then,
they were incubated at 100 °C in an oven in the air overnight to seal the
glass−carbon junction. Each electrode tip was examined and cut close to
the carbon−glass junction using a scalpel under a microscope. To achieve
a glass-sealed disc carbon surface, a micropipette beveler (model BV-10,
Sutter Instrument Co., Novato, USA) was used to polish the tip surface at
45°. Only electrodes that achieved a steady state current in cyclic
voltammetry measurements were used for later sensor fabrication via tip
surface modification.

2. Cyclic Voltammetry: Cyclic voltammetry measurements were conducted

by placing carbon electrodes (backfilled with 3 M KCl solution and a
tungsten wire as a connection) into 1 mM FcMeOH versus a saturated
Ag/AgCl reference electrode (CH Instruments, USA) and scanning at 0.1
V s−1 between −0.2 and +0.8 V using a potentiostat (model 650A Series
Multi-Potentiostat, CH Instruments, USA).

3. Electrode Surface Coating: The electrode tip surface was coated with 20
nm diameter gold nanoparticle (AuNP) hemispheres via electrochemical
deposition, as reported in a recent study. The surface area of the deposited
AuNP was electrochemically determined by first obtaining the surface
charge of the AuNP via a linear sweep at a rate of 0.1 V s−1 from +1.4 V
 to +0.5 V in 500 mM H2SO4, integrating the resulting peak at
approximately +0.8 V, and then dividing the Au surface charge density
by 489 μC cm−2.

4. GOx Immobilization: Glucose oxidase (GOx) was immobilized onto the

AuNP-coated electrode surface at a molar ratio of GOx/AuNP (8000:1)
via self-adsorption by dipping the tip of the AuNP-coated carbon
electrodes into a freshly prepared GOx solution made with 10 mM
sodium bicarbonate (pH 8.2) for approximately 2 hours at room
temperature. All glucose sensors were used immediately after fabrication.

5. Sensor Response Measurement: After placing the glucose sensor

(working electrode) and a chlorinated Ag wire (reference electrode) into
glucose liposome solutions, the sensor's response speed was measured via
amperometric recordings by holding it at 0 mV for 15 seconds and then
changing the potential down to -500 mV for 3-5 minutes using a HEKA
patch clamp amplifier (EPC 10, HEKA Elektronik, Lambrecht, Germany)
at room temperature. The signal was recorded at 20 kHz and Bessel
filtered at 10 kHz. The amperometric data were analyzed by a program
from Sulzer's group using Igor Pro 6.37 software (WaveMetrics, Lake
Oswego, OR, USA); they were first smoothed to 5 kHz (binomial sm.)
and detected as a responding spike with more than 5 times the standard
deviation of the derivative of the baseline current at 0 mV with respect to
time.