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The Virus BioResistor (VBR) is a biosensor capable of rapid and sensitive detection of small protein disease markers using a simple dip-and-read modality. For example, the bladder cancer-associated protein DJ-1 (22 kDa) can be detected in human urine within 1.0 min with a limit of detection (LOD) of 10 pM. The VBR uses engineered virus particles as receptors to recognize and selectively bind the protein of interest. These virus particles are entrained in a conductive poly(3,4-ethylenedioxythiophene) or PEDOT channel. The electrical impedance of the channel increases when the target protein is bound by the virus particles. But VBRs exhibit a sensitivity that is inversely related to the molecular weight of the protein target. Thus, large proteins, such as IgG antibodies (150 kDa), can be undetectable even at high concentrations. We demonstrate that the electrochemical overoxidation of the VBR's PEDOT channel increases its electrical impedance, conferring enhanced sensitivity for both small and large proteins. Overoxidation makes possible the detection of two antibodies, undetectable at a normal VBR, with a limit of detection of 40 ng/mL (250 pM), and a dynamic range for quantitation extending to 600 ng/mL.

Electrochemistry intersected nanoscience 25 years ago when it became possible to control the flow of electrons through single molecules and nanostructures. Many surprises and a wealth of understanding were generated by these experiments. Professor Nongjian Tao was among the pioneering scientists who created the methods and technologies for advancing this new frontier. Achieving a deeper understanding of charge transport in molecules and low-dimensional materials was the first priority of his experiments, but he also succeeded in discovering applications in chemical sensing and biosensing for these novel nanoscopic systems. In parallel with this work, the investigation of a range of phenomena using novel optical microscopic methods was a passion of his and his students. This article is a review and an appreciation of some of his many contributions with a view to the future.

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

The author list contained an error: Gregory A. Weiss was not identified as a corresponding author. Both of the corresponding authors, Gregory A. Weiss and Reginald M. Penner, are indicated in this Addition and Correction.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

A polymer-based electrode capable of specific detection of human serum albumin, and its glycated derivatives, is described. The sensor is constructed from a glass microscope slide coated with a synthesized, polythiophene film bearing a protected, iminodiacetic acid motif. The electrode surface is then further elaborated to a functional biosensor through deprotection of the iminodiacetic acid, followed by metal-affinity immobilization of a specific and high-affinity, albumin ligand. Albumin was then quantified in buffer and synthetic urine via electrochemical impedance spectroscopy. Glycated albumin was next bound to a boronic acid-modified, single-cysteine dihydrofolate reductase variant to quantify glycation ratios by square-wave voltammetry. The platform offers high sensitivity, specificity, and reproducibility in an inexpensive arrangement. The detection limits exceed the requirements for intermediate-term glycemic control monitoring in diabetes patients at 5 and 1 nM for albumin and its glycated forms, respectively.

Nb2O5 is a Li+ intercalation metal oxide that is of current interest for lithium ion battery electrodes. The electrophoretic deposition (ED) of Nb2O5 thin-films from aqueous, NbOx colloidal solutions is reported here. For films ranging in thickness from 38 to 144 nm, the mass loading of Nb2O5 on the electrode is correlated with the coulometry of ED using quartz crystal microbalance gravimetry. Crystalline, phase pure films of orthorhombic, T-Nb2O5, are obtained by postdeposition calcination. These films exhibit unusually high specific capacities for Li+-based energy storage as a consequence of ≈70% porosity. For example, a 60 nm thick film displays a specific capacity, Csp, of 420 mAh/g at 5 A/g and 220 mAh/g at 50 A/g, which can be compared with the theoretical Faradaic capacity of 202 mAh/g. T-Nb2O5 films also have a specific energy density in the range from 770-486 Wh/kg, and a specific power density in the range from 9 to 90 kW/kg. These excellent energy storage metrics are attributed to augmentation of the Faradaic capacity by high double-layer capacities enabled by the mesoporous structure of these films.

The virus bioresistor (VBR) is a chemiresistor that directly transfers information from virus particles to an electrical circuit. Specifically, the VBR enables the label-free detection of a target protein that is recognized and bound by filamentous M13 virus particles, each with dimensions of 6 nm ( w) × 1 μm ( l), entrained in an ultrathin (∼250 nm) composite virus-polymer resistor. Signal produced by the specific binding of virus to target molecules is monitored using the electrical impedance of the VBR: The VBR presents a complex impedance that is modeled by an equivalent circuit containing just three circuit elements: a solution resistance ( Rsoln), a channel resistance ( RVBR), and an interfacial capacitance ( CVBR). The value of RVBR, measured across 5 orders of magnitude in frequency, is increased by the specific recognition and binding of a target protein to the virus particles in the resistor, producing a signal Δ RVBR. The VBR concept is demonstrated using a model system in which human serum albumin (HSA, 66 kDa) is detected in a phosphate buffer solution. The VBR cleanly discriminates between a change in the electrical resistance of the buffer, measured by Rsoln, and selective binding of HSA to virus particles, measured by RVBR. The Δ RVBR induced by HSA binding is as high as 200 Ω, contributing to low sensor-to-sensor coefficients-of-variation (

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.

The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4-ethylenedioxy thiophene) or PEDOT. These virus particles, engineered to selectively bind HSA, serve as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provides electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 μL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Zre signal within 15 min. HSA concentrations in the range from 100 nM to 5 μM are detectable. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient-of-variance (COV) ranging from 2% to 8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

Nanoscience and nanotechnology are poised to contribute to a wide range of fields, from health and medicine to electronics, energy, security, and more. These contributions come both directly in the form of new materials, interfaces, tools, and even properties as well as indirectly by connecting fields together. We celebrate how far we have come, and here, we look at what is to come over the next decade that will leverage the strong and growing base that we have built in nanoscience and nanotechnology.

Virus electrodes address two major challenges associated with biosensing. First, the surface of the viruses can be readily tailored for specific, high affinity binding to targeted biomarkers. Second, the viruses are entrapped in a conducting polymer for electrical resistance-based, quantitative measurement of biomarker concentration. To further enhance device sensitivity, two different ligands can be attached to the virus surface, and increase the apparent affinity for the biomarker. In the example presented here, the two ligands bind to the analyte in a bidentate binding mode with a chelate-based avidity effect, and result in a 100 pM experimentally observed limit of detection for the cancer biomarker prostate-specific membrane antigen. The approach does not require enzymatic amplification, and allows reagent-free, real-time measurements. This article presents general protocols for the development of such biosensors with modified viruses for the enhanced detection of arbitrary target proteins.

The separate fields of conducting polymer-based electrochemical sensors and virus-based molecular recognition offer numerous advantages for biosensing. Grafting M13 bacteriophage into an array of poly (3,4-ethylenedioxythiophene) (PEDOT) nanowires generated hybrids of conducting polymers and viruses. The virus incorporation into the polymeric backbone of PEDOT occurs during electropolymerization via lithographically patterned nanowire electrodeposition. The resultant arrays of virus-PEDOT nanowires enable real-time, reagent-free electrochemical biosensing of analytes in physiologically relevant buffers.

Surface patterns of single-stranded DNA (ssDNA) consisting of nanoscale lines as thin as 40 nm were fabricated on polymer substrates for nanotechnology and bioaffinity sensing applications. Large scale arrays (with areas up to 4 [cm.sup.2]) of ssDNA 'nanolines' were created on streptavidin-coated polymer (PDMS) surfaces by transferring biotinylated ssDNA from a master pattern of gold nanowires attached to a glass substrate. The gold nanowires were first formed on the glass substrate by the process of lithographically patterned nanowire electrodeposition (LPNE), and then 'inked' with biotinylated ssDNA by hybridization adsorption to a thiol-modified ssDNA monolayer attached to the gold nanowires. The transferred ssDNA nanolines were capable of hybridizing with ssDNA from solution to form double-stranded DNA (dsDNA) patterns; a combination of fluorescence and atomic force microscopy (AFM) measurements were used to characterize the dsDNA nanoline arrays. To demonstrate the utility of these surfaces for biosensing, optical diffraction measurements of the hybridization adsorption of DNA-coated gold nanoparticles onto the ssDNA nanoline arrays were used to detect a specific target sequence of unlabeled ssDNA in solution. 10.1021/ac100362u

Parallel arrays of either Au or Pd nanowires were fabricated on glass substrates via the electrochemical process of lithographically patterned nanowire electrodeposition (LPNE) and then characterized with scanning electron microscopy (SEM) and a series of optical diffraction measurements at 633 nm. Nanowires with widths varying from 25 to 150 nm were electrodeposited onto nanoscale Ni surfaces created by the undercut etching of a photo-resist pattern on a planar substrate. With the use of a simple transmission grating geometry, up to 60 diffraction orders were observed from the nanowire gratings, with separate oscillatory intensity patterns appearing in the even and odd diffraction orders. The presence of these intensity oscillations is attributed to the LPNE array fabrication process, which creates arrays with alternating interwire spacings of distances d +[DELTA] and d -[DELTA], where d = 25 [micro]m and the asymmetry A varied from 0 to 3.5 [micro]m. The amount of asymmetry could be controlled by varying the LPNE undercut etching time during the creation of the nanoscale Ni surfaces. The Fourier transform of a mathematical model of the nanowire array was used to predict the diffraction intensity patterns and quantitatively determine A for any grating. Additional sensitivity and an expanded diffraction order range were obtained through the use of external reflection (ER) and total internal reflection (TIR) diffraction geometries.

Electrochemical impedance spectroscopy is used to detect the binding of a 148.2 kDa antibody to a 'covalent virus layer' (CVL) immobilized on a gold electrode. The CVL consisted of M13 phage particles covalently anchored to a 3 mm diameter gold disk electrode. The ability of the CVL to distinguish this antibody ('p-Ab') from a second, nonbinding antibody ('n-Ab') was evaluated as a function of the frequency and phase of the measured current relative to the applied voltage. The binding of p-Ab to the CVL was correlated with a change in the resistance, reducing it at low frequency (1-40 Hz) while increasing it at high frequency (2-140 kHz). The capacitance of the CVL was virtually uncorrelated with p-Ab binding. At both low and high frequency, the electrode resistance was linearly dependent on the p-Ab concentration from 20 to 266 nM but noise compromised the reproducibility of the p-Ab measurement at frequencies below 40 Hz. A 'signal-to-noise' ratio for antibody detection was computed based upon the ratio between the measured resistance change upon p-Ab binding and the standard deviation of this change obtained from multiple measurements. In spite of the fact that the impedance change upon p-Ab binding in the low frequency domain was more than 100 times larger than that measured at high frequency, the S/N ratio at high frequency was higher and virtually independent of frequency from 4 to 140 kHz. Attempts to release p-Ab from the CVL using 0.05 M HCI, as previously described for massbased detection, caused a loss of sensitivity that may be associated with a transition of these phage particles within the CVL from a linear to a coiled conformation at low pH.

M13 virus particles were covalently attached to a planar gold-coated quartz crystal microbalance (QCM) through reaction with a self-assembled monolayer of N-hydroxysuccinimide thioctic ester, followed by incorporation of the blocking agent bovine serum albumin. This immobilization chemistry produced a phage multilayer having a coverage equivalent to [approximately equal to] 6.5 close-packed monolayers of the virus. The properties of this 'covalent virus surface' or CVS for the mass-based detection of a 148.2 kDa antibody were then evaluated in a phosphate buffer using a flow injection analysis system. The mass of the CVS increased with exposure to an antibody (p-Ab) known to bind the phage particles with high affinity. Bound p-Ab was removed by washing with 0.5 M HCI thereby regenerating the sensor surface. A calibration plot for p-Ab binding was constructed by repetitively exposing the surface to p-Ab at concentrations between 6.6 and 200 nM and HCl rinsing after each exposure. The mass--concentration relationship was linear with a sensitivity of 0.018 [micro]g/([cm.sup.2] nM) and a limit of detection of 7 nM or 1.3 pmol. The CVS could be saturated with high doses of p-Ab enabling the determination that an average of [approximately equal to] 140 binding sites are available per M13 phage particle. Exposure of the CVS to a second, nonbinding antibody (n-Ab) did not cause a measurable mass change. These results demonstrate that the covalent virus layer is a rugged, selective, and sensitive means for carrying out mass-based biodetection.

A dense virus layer, readily tailored for recognition of essentially any biomarker, was covalently attached to a gold electrode surface through a self-assembled monolayer. The resistance of this 'virus electrode', [Z.sub.Re], measured in the frequency range from 2 to 500 kHz in a salt-based pH 7.2 buffer, increased when the phage particles selectively bound either an antibody or prostate-specific membrane antigen (PSMA), a biomarker for prostate cancer. In contrast to prior results, we show the capacitive impedence of the virus electrode, [Z.sub.Im], is both a noisier and a less sensitive indicator of this binding compared to [Z.sub.Re]. The specificity of antibody and PSMA binding, and the absence of nonspecific binding to the virus electrode, was confirmed using quartz crystal microbalance gravimetry.