UNLOCKING THE POWER OF NANOPORES

ABSTRACT

Biomedical innovation and the creation of new technology have always been encouraged in this
discipline. The need for picoampere-level current detection in biomedicine has grown since the
turn of the century, driving ongoing advancements in biosensor technology. Nanopore sensing is
one of the most promising new biological sensing technologies. Applications of nanopore
sensing, including chiral compounds, DNA sequencing, and protein sequencing, are reviewed in
this study. However, the detection bandwidths and ionic currents for various compounds range
considerably from one another. In light of this, this article concentrates on current sensing
circuits and presents the most recent circuit architectures and design approaches for various
transimpedance amplifier feedback components, which are mostly utilized in nanopore DNA
sequencing.

INTRODUCTION

When biomolecules like DNA, proteins, and peptides interact with nanopores, a form of
biosensor known as a nanopore sensor—which is built on microscopic pores—can identify and
analyze the biomolecules. In order to detect molecules by their interactions with the orifice, such
as translocating, partially entering, or molecular interactions via particular tags, nanopore sensors
typically have a tiny orifice and two electrodes. The size, shape, and other characteristics of the
molecules cause varied effects when they interact with the pore, resulting in distinct signals that
may be used to ascertain the characteristics and concentrations of the molecules. Numerous
domains, including as DNA sequencing, protein sequencing, chiral molecule discrimination, and
the identification of heavy atoms in non-biological substances, have found extensive uses for
nanopore sensors. Commercial sequencers based on developed biological nanopores have shown
promise for using nanopores. However, issues including amino acid discrimination, translocation
control, and spatial structure unfolding provide difficulties for protein sequencing. There are
several obstacles in the way of using nanopores in the biosensing sector. For example, proteins
made up of 20 natural amino acids could need specifically made nanopores that are the same size
as the smallest units of the molecule. More significantly, the nanopore's detecting zone must be
precisely engineered to offer the necessary sensitivity for the ionic current produced by the
 smallest identifiable units. Because of the limits of current engineering techniques, it is required
to investigate novel approaches in order to modify the shape and size of nanopores, such as de
novo nanopore design and DNA origami scaffolds. In the present research, we have examined
and analyzed solid-state and biological nanopores, as well as nanopore applications in protein
sequencing, chiral compounds, and DNA sequencing. Additionally, we have compiled and
discussed the state-of-the-art transimpedance amplifier design for nanopore simulation front-end
circuits based on the categorization of feedback components. A specific current amplification
circuit, often an integrated circuit, is utilized to detect the ionic current generated by nanopores.
These currents usually have an amplitude at the pA level. Therefore, a high-gain transimpedance
amplifier (TIA) is used at the front end of the application-specific integrated circuit (ASIC) used
for nanopore sensing. The TIA creates a certain circuit architecture by connecting to feedback
components, which enables the conversion of current to voltage. Resistive feedback and
capacitive feedback are the two basic categories into which feedback elements fall. The
transimpedance gain for resistive feedback is the same as the resistance value. High gain and low
input reference noise are necessary for nanopore current amplification, so a significant feedback
resistance—typically in the giga-ohms (GΩ) range—is needed.

ADVANTAGES
1. Only nanopore sequencing provides real-time analysis (for quick insights), completely scalable
formats, the ability to analyze raw DNA or RNA, and the ability to sequence any length of
fragment to produce short to ultra-long read lengths.
2. Applications for nanopore sequencing include real-time analysis, control over the time-to-results,
mobility, and ease of setup. A range of input materials, including natural RNA, cDNA,
amplified DNA, and genomic DNA, have been used to demonstrate the method.
DISADVANTAGES
1. The reason for low resolution is because the DNA strand passes through the nanopore quickly,
moving at a pace of of 1 to 5μs per base. As a result, single nucleotide resolution cannot be
obtained and recording becomes challenging and noisy.
APPLICATIONS OF NANOPORE
 Sequencing of DNA
The foundational method for genetic analysis is DNA sequencing technology, which is crucial to
the diagnosis and management of diseases in the present generation of gene-based human
medical technology. The field of DNA sequencing technology is constantly evolving. Sanger
sequencing, next-generation sequencing (NGS), and the most recent third-generation single-
molecule sequencing technology are the three generations of DNA sequencing advancement.
Scholars have dubbed nanopore sequencing the fourth-generation sequencing technique because
of the tremendous advancement it represents in the field. When an unravelled nucleic acid strand
travels through a nanopore, nanopore sequencing—as opposed to single-molecule real-time
sequencing (SMRT) and Heli-Scope single-molecule sequencing technology—detects extremely
minute variations in current. It is moving in the direction of cheap cost, extended read durations,
and high throughput . A tiny protein pore inserted in an electrically resistant polymer membrane
serves as a biosensor in nanopore sequencing. Negatively charged single-stranded DNA
molecules are driven from the negative "cis" side to the positive "trans" side of the nanopore by a
steady voltage. Base identification accuracy is quite low due to the difficulty of distinguishing
base information from noise caused by the rapid passage of free DNA molecules through the
nanopores. In order to control and ratchet the nucleotides through the nanopore base-by-base and
modulate translocation speed, researchers use DNA polymerases or helicases. The ionic current
flowing through a nanopore is influenced by the characteristics of the nucleobases since the
sensing area of the nanopore is situated at its smallest aperture.

 Sequencing of Proteins

Research on nanopores is moving quickly in the direction of protein sequencing and analysis.
Within the next ten years, nanopore-based protein sequencing might be commercialized.
Unfortunately, mass spectrometry and Edman degradation which have been around for 20 years,
are the current technologies used in protein sequencing and cannot achieve single-molecule
resolution. Although mass spectrometry has emerged as the preferred method for protein
sequencing, there are several limitations with regard to its dynamic detection range and detection
sensitivity. As an alternative, single-molecule resolution protein sequences may be read using
nanopore-based protein sequencing techniques, giving protein sequencing technology fresh life.
By differentiating between the four bases travelling through the sensing area, nanopore-based
DNA sequencing produces an electric current. However, because proteins are biological
macromolecules with special properties, there are three challenges that must be solved when
using them for protein sequencing. First, the biggest challenge in creating a nanopore protein
sequencer is controlling peptide translocation. The charge distribution of amino acid residues
varies greatly from that of evenly charged DNA molecules. Consequently, it is not practical to
employ electrophoretic-driven peptides for straightforward unidirectional translocation via the
nanopore. Second, one must take into account unfolding structured proteins. Most proteins fold
in their original state, and their secondary and tertiary structures need to be broken in order for
them to effectively undergo translocation via the nanopore.

 Chiral Molecules

Beyond DNA sequencing, nanopore technology has many other uses; chiral molecule
identification is one exciting new area of use. Chirality is an essential element of the biological
and chemical domains, a fascinating and complex facet of both sciences. Chromatography and
spectroscopy are two common techniques for chiral separation and identification. But new
biosensing techniques using nanopore technology are starting to make their way into this
industry.

By creating particular chiral environments, single-molecule methods based on nanopores have

demonstrated promise in the sensing of chiral compounds. Boersma et al. created a novel
technique to discriminate between enantiomers of underivatized amino acids using real-time
protein nanopores. According to their research, enantiomers' structural and chemical
characteristics might be translated into unique ionic current signals using nanopores.
Furthermore, an inherently chiral protein nanopore has been described by Cooper et al. to
function as a detecting element to distinguish the chirality of otherwise similar supramolecular
entities. These methods haven't been used, yet, for detecting individual amino acids inside
individual peptides. In this field, more investigation is required.

NANOPORES OVERVIEW
  Biological Nanopores

Despite David Deamer's 1989 notebook proposal, the concept of using nanopores for molecular
detection did not garner much attention until Kasianowicz et al.'s 1996 demonstration of DNA
translocation detection via α-hemolysin. Transporting molecules across cell membranes via
channel proteins served as the impetus for this discovery, which has since illuminated
contemporary nanopore technology. When nanopore research first began, the majority of pores
were intrinsic to channel proteins like α-hemolysin (α-HL), MspA and the channel protein of the
bacteriophage phi29. In a recent study of single-molecule biological nanopore sensing, Mayer et
al. outlined sixteen biological nanopores that are utilized for nanopore sensing, providing a brief
overview of each nanopore's characteristics or uses. Apart from these sixteen biological
nanopores, new applications for nanopore sensing have made use of the biological nanopores
CytK and YaxAB. The primary benefit of these nanopores is their very distinct pore topologies,
which are amenable to manipulation through sophisticated engineering methods enabling
modifications to many elements of the pore's activity.These modifications include adding
reactive amino acids or hydrophobic groups to improve organic molecule binding, or changing
the amount of charges in the channel. Moreover, one benefit of biological nanopores is that the
surface charge of amino acids within the nanopore may be adjusted by varying the pH, which
leads to modifications in electroosmotic flow.

 Solid-state nanopores

Solid-state nanopores are becoming more and more popular due to their great stability and
potential for large-scale manufacture. Nanopore manufacturing has been effectively applied to a
variety of materials, including silicon , silicon nitride, silicon oxide , polymers , aluminium oxide
, and graphene. Phosphorene, MoS2 , and MXene have all been proven to be useful in solid-
state nanopore creation and suitable for DNA detection in recent investigations. Creating a
nanopore often entails opening and fine-tuning processes using tools like FIB drilling, e-beam
drilling, plasma etching, and wet etching. To obtain the appropriate pore size and shape, further
modifications like deposition and heat treatment are typically required. Furthermore, in order to
obtain the appropriate pore size and shape, other modifications like deposition and heat treatment
are typically required. Nanopores with different dimensions, forms, and properties can be
produced by combining these methods. The adoption of silicon nitride-based solid-state
nanopores, for instance, has been greatly aided by the use of inorganic materials and
microfabrication techniques. These nanopores offer high stability and can function under a
variety of experimental conditions, such as variations in voltage, temperature, pressure, pH, and
ionic strength.

 Design of Transimpedance Am.plifier

The flexibility of the transresistance amplifier design is enhanced by the feedback components'
adaptability. This chapter presents state-of-the-art designs for each feedback situation and classes
transresistance amplifiers according to the feedback components (Figure 1). We also offer
insightful guidance to help with the design process.

(Figure 1)

Resistive Feedback

Standard resistors, which serve as the feedback element for TIAs, need an enormous resistance
value of 1 GΩ in order to achieve the desired amplification factor for measurement. This results
in significant thermal noise, a limitation of the circuit bandwidth due to the unavoidable parasitic
capacitance, and an enormous area requirement during chip layout.

 Pseudo Resistance

Because pseudo-resistors can offer adjustable high-value resistance and are a very useful
substitute for ohmic resistors, they have cemented their unique place in the biomedical sensing
industry. By connecting MOS transistors to diodes that exhibit resistive behavior in weak reverse
areas, they have emerged as a crucial technological tool for circuit designers. However, engineers
have suggested new ways to enhance pseudo-resistor designs because of their vulnerability to
process and temperature fluctuations.

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For instance, Djekic et al. connected many pseudo-resistor components in series to greatly increase the
linearity of the transimpedance characteristic [131] and employed a pseudo-current mirror (P-CM) [130]
to lessen the impact of process, voltage, and temperature (PVT) fluctuations. Finally, in order to obtain
the maximum noise level that Ohmic resistance may produce, the suggested enhanced Multi-Element
Pseudo-Resistor (MEPR) was employed as a feedback element for the resistive feedback TIA. Because of
this, it's now a useful technique for creating high-performance TIA for cutting-edge uses like nanopore
biosensing. According to Kim et al. [122], variations in the command voltage required to regulate DNA
mobility during sensing cause the conventional pseudo-resistor approach to have variable pseudo-
resistor values when used for nanopore current sensing [132]. To solve this problem, they suggested
building pseudo-resistor TIAs using a gate-source voltage (VGS)-constant pseudo-resistor approach.
Nonetheless, the MEPR technique, which is appropriate for nanopore biosensing applications, depends
proportionately on the reference resistor and is independent of other variables [131. With the advent of
multi-element pseudo-resistor technology, preamplifier designs may now be tuned to achieve a high
dynamic range. Djekic et al. [15] used a dominating feedback capacitor in the R-TIA design in 2021 and
implemented all resistors as MEPRs. This meant that the transimpedances of both AC and DC could be
adjusted over a period of fifty years, from 440 kΩ to 150 GΩ. They used adjustable multi-element
pseudo-resistors (MEPRs) on an I-D-TIA design a year later [14]. They were able to tune AC and DC
transimpedances between 3 MΩ–1 GΩ and 460 kΩ–300 GΩ, respectively, and achieve a dynamic range
of input currents spanning over five decades by lowering leakage current in the biasing network and
putting enhanced ESD protection mechanisms into place. There may be more uses for improved
amplifiers with longer dynamic range in biosensing.

4.1.2. Switched Resistors

Owing to their high degree of flexibility in resistance values and arrangements, switched resistors are
widely used in a variety of circuit designs for biomedical applications. For instance, they can be utilized
as essential components to accomplish various goals in active RC filters [133], preamplifiers [134],
switched resistor filters [135,136], pulse generators [137], and other circuits. Additionally, because CMOS
technology is so widely used, switched resistors are used for analogue signal processing in digital circuits.
For instance, employing switched resistors in digital-to-analog converters (DACs) can lessen the effect of
reference path resistance on DAC performance [138].

Switched resistors, however, were not used as feedback components in transimpedance amplifiers for
biological applications until just [16]. A transmission gate switch (TG) and a polysilicon resistor are used
in series in the traditional switched resistor technique [16,135], with the control provided by a digitally
programmable clock signal. The equivalent conductance of the circuit changes in tandem with changes in
the average current flow through the series and the clock signal's duty cycle. Because the conductance is
related to the clock signal's duty cycle, digital control signals may be used to easily alter the circuit
conductance. Due to TIA's low noise front-end, Centurelli et al. [16] Also, they reduced the noise and
power consumption of pseudo-resistors in simulations by optimizing the design of the Operational
Transconductance Amplifier (OTA) to obtain low input noise.

Future studies may find value in investigating the distributed switching resistor technique [135] for TIA
design. Even with little duty cycles, this method can reach high resistance multiplication factors of
several thousand. As a result, it successfully lessens the detrimental impacts of parasitic capacitances on
equivalent resistance values, which is crucial for TIA since higher gain is required.

4.1.3. Ohmic-Resistor

The resistance value of an ohmic resistor is directly proportional to the current flowing through it, and it
is characterized by a linear connection with respect to Ohm's law. These resistors fall into three
categories: poly, chip, and through-hole. They are all often employed in circuits to supply accurate,
consistent, and dependable resistance values. On the other hand, large parasitic capacitance and
considerable thermal noise can be caused by high resistance levels. Furthermore, direct integration is
not feasible due to the enormous chip surface occupied by high-resistance polyresistors [13,131].
However, techniques like parasitic resistance reduction and noise reduction have been proposed to
enable the application of this feedback method in biosensor interface circuits for nanopores and other
applications, because of their simpler circuit structure than minimizing capacitor feedback and pseudo-
resistor feedback [139, 140, 141].

First, Gu et al. [139] found that noise in nanopore sensing may be significantly minimized by decreasing
membrane capacitance, which amplifies noise from applied voltage. Based on resistive feedback TIA
technology, they created an instrumentation system with ultra-low noise performance that records
single-molecule signals with a p-p noise of 3.26 pA when using a 5 kHz filter. Furthermore, to overcome
the major drawback of large parasitic capacitance coming from utilizing high feedback resistance on the
3 dB bandwidth, Yun et al. used a unique approach termed a split-resistor [140]. The split-resistor
method decreases the parasitic capacitance of each resistor by around N times by splitting a big resistor
into N equal resistance sections. Consequently, the 3 dB bandwidth expanded from 70 Hz to 41 kHz
while maintaining the benefits of high feedback resistance-provided high gain of 179.9 dBΩ and low
input noise of 570 fARMS. Furthermore, Zhong et al. used a circuit board with amplifiers and an array of
Au electrodes to produce a four-channel electrochemical instrument with low noise level. A two-stage
amplifier with a frequency correction circuit powers their device [142]. High bandwidth and low noise
performance are made possible by the ultra-low picoampere-level current being amplified to the
millivolt level with a current gain of 180 dB without the need for GΩ-level feedback resistors, thanks to
the first- and second-stage amplifiers' gains of 160 dB and 20 dB, respectively. CMOS designs have also
utilized this cascaded amplification technique.

All three of the above mentioned investigations were conducted in a distinct manner. We now turn our
attention to CMOS technology application. A low-noise readout circuit with a bandwidth of up to 1.3
MHz and a noise amplitude as low as 11 fARMS/sq(Hz) was proposed by Wang et al. [141]. Because of
the innovative electrostatic discharge (ESD) leakage current cancellation step, the circuit can detect
current inputs in the sub-pA range. A preamplifier with a current gain of 40 dB is used to amp up the
current, and a TIA with a feedback element of a 1 MΩ Poly resistor is used as a post-stage amplifier to
provide a total transmission impedance gain of 160 dB. The way that CMOS circuit design is approached
has changed. In order to deliver the necessary transresistance gain, designers are no longer depending
only on feedback resistors due to their limits in obtaining the requisite gain level. In order to get the
front-end amplification circuit's desired overall gain, a multi-stage amplification technique has been used
instead.

In a similar vein, Kim and colleagues have shown a low-power, high-precision interface circuit for DNA
reading in multiple nanopore applications [143]. They decided to employ a resistive feedback TIA and a
voltage gain non-inverting amplifier at the TIA's output stage to make up for inadequate transimpedance
gain because of the hardware's dependability and simplicity. In order to eliminate the requirement for an
output buffer, they suggested replacing the differential amplifier with a non-inverted structure.
Additionally, they suggested adding a new offset cancellation module to minimize detrimental input
offset voltage and greatly increase accuracy. Traditional resistive feedback TIA circuits are more suited for
applications because of the hardware's reliability and simplicity, which free up circuit designers to
concentrate on improving other parts of the circuit.

4.2. Capacitive Feedback

The goal of TIA design in biosensing has always been to achieve low noise and high signal-to-
noise ratio since physiological signals are readily overpowered by noise. Because capacitors are
noiseless components that improve noise performance in TIA design, they are a good fit for
capacitive feedback elements. Capacitive feedback is therefore growing in popularity. The
output voltage in a capacitive feedback architecture is the result of the input current integrated.
Capacitive feedback TIA does, however, have a serious drawback: the charge that builds up
across the capacitor may cause the amplifier's output voltage to become saturated. There are
two main methods for getting around this restriction. The first approach is a discrete-time (DT)
methodology that adds a reset switch on a regular basis to release the charge held in the
feedback capacitor. The continuous-time (CT) technique is an additional strategy that prevents
output saturation by removing the baseline current through the addition of a DC cancellation
circuit.

4.2.1. Discrete-Time Capacitive Feedback

Figure 4A displays the schematic design of a conventional discrete-time reset model [144]. To
avoid operational amplifier saturation, the feedback capacitor is periodically reset. Resetting,
however, may cause data loss, especially at lower frequencies when it's important to reduce
reset time. Using a lower feedback capacitor—typically between 15 and 50 fF—will increase
sensitivity. An analytical model for switched-capacitor transimpedance amplifiers (SCTIAs) was
recently proposed by Jiang et al. [145]. Their transimpedance amplifier (TIA) was constructed
utilizing cross-coupling and correlated double sampling (CDS) (Figure 5) in order to improve the
transimpedance gain folded back to the input, noise current, and power consumption. Their
model was based on charge redistribution. The TIA was able to attain a 3 kHz bandwidth and
206 dB transimpedance gain. At 1 kHz, the baseline noise was 2.96 fA/√Hz, and 0.643 mW of
power were used. A completely differential amplifier and several logic-controlled MOS switch
toggles are used in the SCTIA model. Voltage sampling is finished and the saturation issue of the
feedback capacitor is resolved by adjusting the time of three pairs of switches. With the use of
the CDS approach, offset and flicker noise may be completely removed from the output in this
design. KCT noise and folded-back high-frequency noise in the front-end circuit usually impair
the DT reset technique. To improve the readout sensitivity, the correlated double sampling
(CDS) approach is used as a remedy [146,147]. In order for CDS to function, the input signal
must be sampled twice. The first sample comprises only low-frequency noise and offset, while
the second sample includes both the signal and the low-frequency noise together.
Figure 4. (A) Feedback network of discrete-time TIA and (B) feedback network of
continuous-time TIA [144]. Reproduced with permission from Liu, Fan, Chen, Wan, Mao,
and Yu, Front. Electron., published by Frontiers Media, 2022.

Figure 5. A typical front-end architecture utilizes the CDS-based discrete-time method

[144]. Reproduced with permission from Liu, Fan, Chen, Wan, Mao, and Yu, Front.
Electron., published by Frontiers Media, 2022

Compared to conventional DT-TIA circuits, the hourglass ADC method used by Hsu and Hall
[148] offers a completely new design for signal amplification and digitization. The polarity of the
input signal can be reversed within a user-defined time window by using an hourglass switch in
place of regularly resetting the TIA feedback capacitor. This increases sensitivity and linearity
and solves the issue of saturation in the integrator stage. Additionally, the hourglass ADC
transforms the integrator output from voltage to frequency, enabling a great dynamic range. As
a result, this innovative method could stimulate fresh conceptualization and encourage the
creation of DT-TIAs that are more effective and perform better.

Achieving high throughput is critical for DNA sequencing technology. To address this
need, Dawji et al. have developed an amplifier array capable of reading high-speed
mixed signals using 130-nm CMOS technology [149]. This array amplifies and digitizes
picoampere-range current signals. It comprises thirty identical channels that work in
tandem, with each channel featuring a DT-TIA and an in-pixel SAR ADC operating with
correlated double sampling (CDS). The DT-TIAs can detect nanopore currents at the
picoampere level, offering a gain of G ohm, which represents the first time a complete,
multiple-channel, integrated ADC array ROIC has been reported for nanopore-based
DNA sequencing. Additionally, it consumes only one-tenth of the power usage of other
DT-TIAs.

4.2.2. Continuous-Time Capacitive Feedback

resistant materials, polyethylene glycol (PEG) polymers or zwitterionic polymers, based on the increased
permeability and retention (EPR) effect.Unfortunately, because of PEG's high protein adsorption resistan
ce structure, PEGylation of NDDS negatively affects NDDS internalization, decreasing its therapeutic impa
ct.