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Nanopore sensing is a popular biosensing strategy that is being explored for the quantitative analysis of biomarkers. With low concentrations of analytes, nanopore sensors face challenges related to slow response times and selectivity. Here, we demonstrate an approach to rapidly detect species at ultralow concentrations using an optical nanopore blockade sensor for quantitative detection of the protein vascular endothelial growth factor (VEGF). This sensor relies on monitoring fluorescent polystyrene nanoparticles blocking nanopores in a nanopore array of 676 nanopores. The fluorescent signal is read out using a wide-field fluorescence microscope. Nonspecific blockade events are then distinguished from specific blockade events based on the ability to pull the particles out of the pore using an applied electric field. This allows the detection of VEGF at sub-picomolar concentration in less than 15 min.

Herein the development of cellular impedance biosensors, electrochemical impedance spectroscopy, and the general principles and terms associated with the cell-electrode interface is reviewed. This family of techniques provides quantitative and sensitive information into cell responses to stimuli in real-time with high temporal resolution. The applications of cell-based impedance biosensors as a readout in cell biology is illustrated with a diverse range of examples. The current state of the field, its limitations, the possible available solutions, and the potential benefits of developing biosensors are discussed.

Affinity biosensors play a crucial role in clinical diagnosis, pharmaceuticals, immunology, and other areas of human health. Affinity biosensors rely on the specific binding between target analytes and biological ligands such as antibodies, nucleic acids, aptamers, or other receptors to primarily generate electrochemical or optical signals. Considerable effort has been put into improving the performance of the affinity technologies to make them more sensitive, efficient and reproducible, of the many approaches electrokinetic phenomena are a viable option. In this perspective, studies that combine electrokinetic phenomena with affinity biosensor are discussed about their promise for achieving higher sensitivity and lower detection limit., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Alterations in DNA methylation, a stable epigenetic marker, are important components in the development of cancer. It is vital to develop diagnostic systems with the ability to rapidly quantify DNA methylation with high sensitivity and selectivity. However, the analysis of DNA methylation must address two main challenges: (i) ultralow abundance and (ii) differentiating methylated cytosine from normal cytosine on target DNA sequence in the presence of an overwhelming background of circulating cell-free DNA. Here we report the development of an ultrasensitive and highly-selective electrochemical biosensor for the rapid detection of DNA methylation in blood. The sensing of DNA methylation involves the hybridization on a network of probe DNA modified gold-coated magnetic nanoparticles (DNA-Au@MNPs) complementary to target DNA, and subsequently enzymatic cleavage to differentiate methylated DNA strands from corresponding unmethylated DNA strands. The biosensor presents a dynamic range from 2 aM to 20 nM for 110 nucleotide DNA sequences containing a single-site methylation with the lowest detected concentration of 2 aM. This DNA-Au@MNPs based sensor provides a promising method to achieve 35 min response time and minimally invasive diagnosis of ovarian cancer., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Knowledge of the interaction between aptamer and protein is integral to the design and development of aptamer-based biosensors. Nanoparticles functionalized with aptamers are commonly used in these kinds of sensors. As such, studies into how the number of aptamers on the nanoparticle surface influence both kinetics and thermodynamics of the binding interaction are required. In this study, aptamers specific for interferon gamma (IFN-γ) were immobilized on the surface of gold nanoparticles (AuNPs), and the effect of surface coverage of aptamer on the binding interaction with its target was investigated using fluorescence spectroscopy. The number of aptamers were adjusted from an average of 9.6 to 258 per particle. The binding isotherm between AuNPs-aptamer conjugate and protein was modeled with the Hill-Langmuir equation, and the determined equilibrium dissociation constant ( K ' D ) decreased 10-fold when increasing the coverage of aptamer. The kinetics of the reaction as a function of coverage of aptamer were also investigated, including the association rate constant ( k on ) and the dissociation rate constant ( k off ). The AuNPs-aptamer conjugate with 258 aptamers per particle had the highest k on , while the k off was similar for AuNPs-aptamer conjugates with different surface coverages. Therefore, the surface coverage of aptamers on AuNPs affects both the thermodynamics and the kinetics of the binding. The AuNPs-aptamer conjugate with the highest surface coverage is the most favorable in biosensors considering the limit of detection, sensitivity, and response time of the assay. These findings deepen our understanding of the interaction between aptamer and target protein on the particle surface, which is important to both improve the scientific design and increase the application of aptamer-nanoparticle based biosensor.

A massively parallel single particle sensing method based on core-satellite formation of Au nanoparticles was introduced for the detection of interleukin 6 (IL-6). This method exploits the fact that the localized plasmon resonance (LSPR) of the plasmonic nanoparticles will change as a result of core-satellite formation, resulting in a change in the observed color. In this method, the hue (color) value of thousands of 67 nm Au nanoparticles immobilized on a glass coverslip surface is analyzed by a Matlab code before and after the addition of reporter nanoparticles containing IL-6 as target protein. The average hue shift as the result of core-satellite formation is used as the basis to detect small amount of proteins. This method enjoys two major advantages. First it is able to analyze the hue values of thousands of nanoparticles in parallel in less than a minute. Secondly the method is able to circumvent the effect of non-specific adsorption, a major issue in the field of biosensing., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recent advances in CRISPR based biotechnologies have greatly expanded our capabilities to repurpose CRISPR for the development of biomolecular sensors for diagnosing diseases and understanding cellular pathways. The key attribute that allows CRISPR to be widely utilized is the programmable and highly selective mechanism. In this Minireview, we first illustrate the molecular principle of CRISPR functioning process from sensing to actuating. Next, the CRISPR based biosensing strategies for nucleic acids, proteins and small molecules are summarized. We highlight some of recent advances in applications for in vitro detection of biomolecules and in vivo imaging of cellular networks. Finally, the challenges with, and exciting prospects of, CRISPR based biosensing developments are discussed., (© 2020 Wiley-VCH GmbH.)

The detection principle of nanopore sensors relies on measuring changes in electrical signal as analyte molecules translocate through a nanoscale pore. There are two challenges with this experimental construct when using nanopores for quantitative sensing with low detection limits in complex samples. The first is getting the analyte to the nanopore in a reasonable time frame and the second is other species in the sample also translocating through the nanopore and generating erroneous signals. We have developed a nanopore blockade sensor that alleviates the limitations of diffusion-limited mass transport and non-specific signals. Antibody-modified magnetic nanoparticles are utilized to deliver analytes of interest extracted from sample to an array of antibody-modified nanopores under a controlled electromagnet, resulting in long-term nanopore blocking events due to the formation of sandwiched immunocomplexes. Herein, this study reports on understanding some of important parameters in determining the performance of nanopore blockade sensing system, where prostate-specific antigen (PSA) is used as a model analyte. We describe the characterization of nanopore blockade sensing of PSA by (1) tuning on/off the electromagnet, (2) varying nanopore number in a nanopore chip, and (3) deploying the sensor in human plasma. Results show that magnetophoresis effectively facilitates active delivery and selective sensing of PSA to the nanopore. Nanopore chips with a larger number of nanopores are shown to receive more nanopore blockades for a given concentration of analyte. Furthermore, identifiable blockade events accounted for successful detection of PSA in plasma, indicate the high specificity of the sensing system., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Personal glucose meters (PGMs) have been used for the measurement of blood glucose for decades now such that they have become the most used analytical method in the world. They are also well placed to be repurposed for point-of-care testing of other analytes as they are inexpensive, portable and quantitative. Efforts to repurpose PGMs for the detection of any analyte at the point-of-care have been one focus of biosensor research for several years now with a number of successful efforts in the detection of a wide range of analytes. This article reviews the published methods to repurpose a PGM to detect analytes other than glucose, and analyses the potential and the challenges to be overcome in developing a PGM-based biosensor and bring it to market., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Optical biosensors are defined as portable optical devices that use biorecognition molecules to interrogate a sample for the presence of a target. The capabilities of optical biosensors have expanded rapidly with advances in miniature optical components and molecular engineering. Biosensors to meet the needs in health and environmental monitoring and food safety have become commercially available, with many more in the pipeline. We review the innovative approaches to overcoming existing hurdles to practical biosensor designs and explore potential areas for future breakthroughs in optical biosensor technology.

Nanopore sensors detect individual species passing through a nanoscale pore. This experimental paradigm suffers from long analysis times at low analyte concentration and non-specific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis. Here, we address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). The (anti-PSA)-MNPs are magnetically driven to block an array of nanopores rather than translocate through the nanopore. Specificity is obtained by modifying nanopores with anti-PSA antibodies such that PSA molecules captured by (anti-PSA)-MNPs form an immunosandwich in the nanopore. Reversing the magnetic field removes (anti-PSA)-MNPs that have not captured PSA, limiting non-specific effects. The combined features allow detecting PSA in whole blood with a 0.8 fM detection limit. Our 'magnetic nanoparticle, nanopore blockade' concept points towards a strategy to improving nanopore biosensors for quantitative analysis of various protein and nucleic acid species.

The work presented here describes the development of an optical label-free biosensor based on a porous silicon (PSi) Bragg reflector to study heterogeneity in single cells. Photolithographic patterning of a poly(ethylene glycol) (PEG) hydrogel with a photoinitiator was employed on RGD peptide-modified PSi to create micropatterns with cell adhesive and cell repellent areas. Macrophage J774 cells were incubated to form cell microarrays and single cell arrays. Moreover, cells on the microarrays were lysed osmotically with Milli-Q™ water and the infiltration of cell lysate into the porous matrix was monitored by measuring the red shift in the reflectivity. On average, the magnitude of red shift increased with the increase in the number of cells on the micropatterns. The red shift from the spots with single cells varied from spot to spot emphasizing the heterogeneous nature of the individual cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This Perspective focuses on the latest strategies and challenges for the development of bioanalytical sensors with sub-picomolar detection limits. Achieving sub-picomolar detection limits has three major challenges: (1) assay sensitivity, (2) response time, and (3) selectivity (including limiting background signals). Each of these challenges is discussed, along with how nanomaterials provide the solutions. One strategy to gain greater sensitivity involves confining the sensing volume to the nanoscale, as used in nanopore- or nanoparticle-based sensors, because nanoparticles are ubiquitous in amplification. Methods to improve response time typically focus on obtaining an intimate mixture between the sensor and the sample either by extending the length scale of nanoscale sensors using nanostructuring or by dispersing magnetic nanoparticles through the sample to capture the analyte. Loading nanoparticles with many biorecognition species is one solution to help address the challenge of selectivity. Many examples in this Perspective explore the detection of prostate-specific antigen which enables a comparison between strategies. Finally, exciting future opportunities in developing single-molecule sensors and the requirements to go even lower in concentration are explored.

The integration of plasmonic nanoparticles into biosensors has the potential to increase the sensitivity and dynamic range of detection, through the use of single nanoparticle assays. The analysis of the localized surface plasmon resonance (LSPR) of plasmonic nanoparticles has allowed the limit of detection of biosensors to move towards single molecules. However, due to complex equipment or slow analysis times, these technologies have not been implemented for point-of-care detection. Herein, we demonstrate an advancement in LSPR analysis by presenting a technique, which utilizes an inexpensive CMOS-equipped digital camera and a dark-field microscope, that can analyse the λ max of over several thousand gold nanospheres in less than a second, without the use of a spectrometer. This improves the throughput of single particle spectral analysis by enabling more nanoparticles to be probed and in a much shorter time. This technique has been demonstrated through the detection of interleukin-6 through a core-satellite binding assay. We anticipate that this technique will aid in the development of high-throughput, multiplexed and point-of-care single nanoparticle biosensors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fibrotic diseases are among the most serious health issues with severe burdens due to their chronic nature and a large number of patients suffering from the debilitating effects and long-term sequelae. Collagenase treatment is a nonsurgical option but has limited results. To date, there is no potent noninvasive solution for fibrosis. Part of the reason for this is the lack of appropriate in vitro live cell screening tools to assess the efficacy of new therapeutical agents. Here, we demonstrate the utility of a cell-based electrochemical impedance biosensor platform to screen the efficacy of potential antifibrotic compounds. The platform employs a label-free and noninvasive strategy to detect the progression of fibrosis and the potency of the antifibrotic molecules in real-time. The fundamental principle that governs this novel system is that dynamic changes in cell shape and adhesion during fibrosis can be measured accurately by monitoring the changes in the impedance. This is achieved by growing the cells on a transparent interdigitated indium tin oxide (ITO) electrodes. It was demonstrated by monitoring the efficacy of a model antifibrotic compound, PXS64, on cells collected from patients with Dupuytren's contracture. We confirmed the validity of the developed biochemical impedance biosensor as an tool for in vitro screening of antifibrotic compounds and provided quantitative information on subcellular influences of the examined chemical molecules using correlative microscopy analyses that monitor the average cell area, cell morphology, and the amount and directionality of the deposited extracellular matrix protein collagen and measurement of cytosolic Ca 2+ changes.

Conventional immunosensors require many binding events to give a single transducer output which represents the concentration of the analyte in the sample. Because of the requirements to selectively detect species in complex samples, immunosensing interfaces must allow immobilisation of antibodies while repelling nonspecific adsorption of other species. These requirements lead to quite sophisticated interfacial design, often with molecular level control, but we have no tools to characterise how well these interfaces work at the molecular level. The work reported herein is an initial feasibility study to show that antibody-antigen binding events can be monitored at the single molecule level using single molecule localisation microscopy (SMLM). The steps to achieve this first requires showing that indium tin oxide surfaces can be used for SMLM, then that these surfaces can be modified with self-assembled monolayers using organophosphonic acid derivatives, that the amount of antigens and antibodies on the surface can be controlled and monitored at the single molecule level and finally antibody binding to antigen modified surfaces can be monitored. The results show the amount of antibody that binds to an antigen modified surface is dependent on both the concentration of antigen on the surface and the concentration of antibody in solution. This study demonstrates the potential of SMLM for characterising biosensing interfaces and as the transducer in a massively parallel, wide field, single molecule detection scheme for quantitative analysis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The influence of RNA versus DNA on the performance of electrochemical biosensors where redox-labelled nucleic acid duplexes bend towards the electrode surface has been assessed. Faster electron transfer was observed for duplexes containing RNA, suggesting duplexes with RNA are more flexible. These data are of particular importance for microRNA biosensors.

The adoption of plasmonic nanomaterials in optical sensors, coupled with the advances in detection techniques, has opened the way for biosensing with single plasmonic particles. Single nanoparticle sensors offer the potential to analyse biochemical interactions at a single-molecule level, thereby allowing us to capture even more information than ensemble measurements. We introduce the concepts behind single nanoparticle sensing and how the localised surface plasmon resonances of these nanoparticles are dependent upon their materials, shape and size. Then we outline the different synthetic approaches, like citrate reduction, seed-mediated and seedless growth, that enable the synthesis of gold and silver nanospheres, nanorods, nanostars, nanoprisms and other nanostructures with tunable sizes. Further, we go into the aspects related to purification and functionalisation of nanoparticles, prior to the fabrication of sensing surfaces. Finally, the recent developments in single nanoparticle detection, spectroscopy and sensing applications are discussed.

Considerable attention has been dedicated to developing feasible point-of-care tests for cancer diagnosis and prognosis. An ideal biomarker for clinical use should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. The role of microRNAs (miRNAs) in the regulation of different cellular processes, the unique altered patterns in cancer patients and presence in body fluids in the stable form, points to their clinical utility as blood-based biomarkers for diagnosis, prognosis, and treatment of cancer. Although a variety of selective and sensitive laboratory-based methods are already exist for the detection of circulating miRNA, having a simple, low-cost and rapid assay, which could be routinely used in clinical practice, is still required. Among different approaches that have developed for circulating miRNA detection, biosensors, due to the high sensitivity, ease of use, short assay time, non-toxic experimental steps, and adaptability to point-of-care testing, exhibit very attractive properties for developing portable devices. With this view, we present an overview of some of the challenges that still need to be met to be able to use circulating miRNAs in clinical practice, including their clinical significance, sample preparation, and detection. In particular, we highlight the recent advances in the rapidly developing area of biosensors for circulating miRNA detection, along with future prospects and challenges., (© 2014 Wiley Periodicals, Inc.)

Two different interfaces prepared via UV-hydrosilylation of undecylenic acid and 1,8-nonadiyne on silicon(111) have been explored to develop a robust electrochemical DNA sensor. Electrodes modified with undecylenic acid were found to stably immobilise DNA but could not resist the growth of insulating oxides, whereas 1,8-nonadiyne modified electrodes satisfy both requirements.

Porous silicon photonics is the ideal platform for high sensitivity, high selectivity monitoring of biological molecules in a complex fluidic environment. The potential of this technology was identified almost 15 years ago, however, it has taken considerable advances in porous silicon surface chemistry, photonics, and micro-fabrication to create truly effective devices that can provide new insights into the behaviour of biological systems. In this review we provide a critical assessment of the development of porous silicon optical biosensors from the early demonstrations of affinity based sensing to the current trends in monitoring single cell activity and perspectives in the use of photonic microparticles for biomedical applications.

Organic coatings on electrodes that limit biofouling by proteins but are of sufficiently low impedance to still allow Faradaic electrochemistry to proceed at the underlying electrode are described for the first time. These organic coatings formed using simple aryl diazonium salts present a zwitterionic surface and exhibit good electrochemical stability. The layers represent a low impedance alternative to the oligo (ethylene glycol) (OEG)-based anti-biofouling coatings and are expected to find applications in electrochemical biosensors and implantable electrodes. Two different zwitterionic layers grafted to glassy carbon surfaces are presented and compared to a number of better-known surfaces, including OEG-based phenyl-layer-grafted glassy carbon surfaces and OEG alkanethiol SAMs coated on gold, to allow the performance of these new layers to be compared to the body of work on other anti-biofouling surfaces. The results suggest that phenyl-based zwitterionic coatings are as effective as the OEG SAMs at resisting the nonspecific adsorption of bovine serum albumin and cytochrome c, as representative anionic and cationic proteins at physiological pH, whereas the impedance of the zwitterionic phenyl layers are two orders of magnitude lower than OEG SAMs.

New sensation: A resistance-based biosensor uses gold-coated magnetic nanoparticles (Au@MNPs) functionalized with the antibiotic enrofloxin (see picture; purple), which bind to anti-enrofloxin as analyte (blue). The Au@MNPs can be magnetically assembled between electrodes, and the measured resistance R is a function of analyte concentration., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An electrochemical immuno-biosensor for detecting glycosylated haemoglobin (HbA1c) is reported based on glassy carbon (GC) electrodes with a mixed layer of an oligo(phenylethynylene) molecular wire (MW) and an oligo(ethylene glycol) (OEG). The mixed layer is formed from in situ-generated aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene (FDMA) was attached followed by the covalent attachment of an epitope N-glycosylated pentapeptide (GPP), an analogon to HbA1c, to which an anti-HbA1c monocolonal antibody IgG can selectively bind. HbA1c was detected by a competitive inhibition assay based on the competition for binding to anti-HbA1c IgG antibodies between the analyte in solution, HbA1c, and the surface bound epitope GPP. Exposure of the GPP modified sensing interface to the mixture of anti-HbA1c IgG antibody and HbA1c results in the attenuation of ferrocene electrochemistry due to free antibody binding to the interface. Higher concentrations of analyte led to higher Faradaic currents as less anti-HbA1c IgG is available to bind to the electrode surface. It was observed that there is a good linear relationship between the relative Faradaic current of FDMA and the concentration of HbA1c from 4.5% to 15.1% of total haemoglobin in serum without the need for washing or rinsing steps.

Electrochemical biosensors have witnessed a tremendous growth in nanotechnology and in depth characterisation over the last two decades. In particular, modification of surfaces with self-assembled monolayers (SAMs) has provided a molecular control of the interfaces, which has paved the way to fabricate improved biosensing devices. Here we focus on the important advances of using SAMs in enzyme electrodes and affinity biosensors such as for peptides, DNA and antibodies., (Copyright © 2012 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.)

A simple colorimetric method for the detection of copper ions in water is described. This method is based on the 'click' copper(I)-catalyzed azide-alkyne cycloaddition reaction and its use in promoting the aggregation of azide-tagged gold nanoparticles by a dialkyne cross-linker is described. Nanoparticle cross-linking, evidenced as a colour change, is used for the detection of copper ions. The lowest detected concentration by the naked eye was 1.8 μM, with the response linear with log(concentration) between 1.8-200 μM. The selectivity relative to other potentially interfering ions was evaluated., (This journal is © The Royal Society of Chemistry 2012)

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An immuno-biosensing interface comprising a mixed layer of an oligo(ethylene glycol) (OEG) component, and an oligo(phenylethynylene) molecular wire (MW) is described. The OEG controls the interaction of proteins and electroactive interferences with the surface and the MW allows electrochemical communication to the underlying glassy carbon electrode. The layers are formed from in situ generated-aryl diazonium cations. To the distal end of the MW, a redox probe 1,1'-di(aminomethyl)ferrocene is attached followed by the surface bound epitope (the structural feature the antibody selectively recognizes) to which an antibody would bind. Association or disassociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. X-ray photoelectron spectroscopy, cyclic voltammetry, and square wave voltammetry have been used to characterize the step-wise fabrication of the sensing interface. The influence of the molar ratio of the MW and OEG deposited onto the sensor interface was explored relative to the final sensor sensitivity. Five combinations of MW/OEG 1:0, 1:20, 1:50, 1:75 and 1:100 were tested on sensor sensitivity detection for a model analyte (biotin) free in solution, via a displacement assay. The ratio of 1:50 was found to give the highest sensitivity. At this ratio, good reproducibility (RSD 6.8%) and repeatability (RSD 9.6%) was achieved. This immuno-biosensor provides an intervention free immuno-biosensing platform for agriculture and biomedical samples., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We report the use of copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) to selectively functionalize the internal and external surfaces of mesoporous materials. Porous silicon rugate filters with narrow line width reflectivity peaks were employed to demonstrate this selective surface functionalization approach. Hydrosilylation of a dialkyne species, 1,8-nonadiyne, was performed to stabilize the freshly fabricated porous silicon rugate filters against oxidation and to allow for further chemical derivatization via "click" CuAAC reactions. The external surface was modified through CuAAC reactions performed in the absence of nitrogen-based Cu(I)-stabilizing species (i.e., ligand-free reactions). To subsequently modify the interior pore surface, stabilization of the Cu(I) catalyst was required. Optical reflectivity measurements, water contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) were used to demonstrate the ability of the derivatization approach to selectively modify mesoporous materials with different surface chemistry on the exterior and interior surfaces. Furthermore, porous silicon rugate filters modified externally with the cell-adhesive peptide Gly-Arg-Gly-Asp-Ser (GRGDS) allowed for cell adhesion via formation of focal adhesion points. Results presented here demonstrate a general approach to selectively modify mesoporous silicon samples with potential applications for cell-based biosensing.

In this paper we demonstrate the possibility of modifying porous silicon (PSi) particles with surface chemistry and recognition molecules (antibodies) such that these devices could potentially be used for single-cell identification or sensing. This is achieved by modifying PSi Rugate filters via hydrosilylation with surface chemistry that serves firstly, to protect the silicon surfaces from oxidation; secondly, renders the surfaces resistant to nonspecific adsorption of proteins and cells and thirdly, allows further functionality to be added such as the coupling of antibodies. The surface chemistry remained unchanged after sonication of the PSi to form PSi microparticles. The ability to monitor the spectroscopic properties of microparticles, and shifts in the optical signature due to changes in the refractive index of the material within the pore space, is demonstrated. The particles are shown to remain stable in physiological buffers and human blood for longer than one week. Finally, the modification of the PSi particles with functional antibodies is achieved.

In this article we present an optical biosensor for label-free detection of trace levels of protease activity. The scheme is based on surface functionalized porous silicon optical structures which supports optical Bloch surface modes. The optical structure provides a resonant optical mode for high sensitivity detection and open access of the sensing layer to the target enzyme. Protease detection is based on the digestion of gelatin, covalently attached inside the pore space, resulting in a spectral blue-shift of the optical mode. Monitoring of spatially separated resonant optical modes is used to eliminate optical response from nonspecific adsorption.

From diagnosis of life-threatening diseases to detection of biological agents in warfare or terrorist attacks, biosensors are becoming a critical part of modern life. Many recent biosensors have incorporated carbon nanotubes as sensing elements, while a growing body of work has begun to do the same with the emergent nanomaterial graphene, which is effectively an unrolled nanotube. With this widespread use of carbon nanomaterials in biosensors, it is timely to assess how this trend is contributing to the science and applications of biosensors. This Review explores these issues by presenting the latest advances in electrochemical, electrical, and optical biosensors that use carbon nanotubes and graphene, and critically compares the performance of the two carbon allotropes in this application. Ultimately, carbon nanomaterials, although still to meet key challenges in fabrication and handling, have a bright future as biosensors.

The ease of fabricating high quality photonic crystals from porous silicon and its biocompatibility have inspired the conception of various biosensing schemes using this material. However, the instability of porous silicon has significantly slowed progress in this area. Here we discuss the potential of different porous silicon photonic crystals for biosensing in the context of its surface chemistry and nanostructure, both of which need to be optimized to obtain sensitive and stable devices. Of particular promise are recent approaches that use porous silicon as sensors for enzymatic activity, for cell capture and concentration devices.

Herein we present a label-free immunobiosensor based on the modulation of amperometric signals of surface bound redox species when immersed in a protein environment which is applicable to either the detection of antibodies or the detection of small molecules such as drugs or pesticides.

The organic derivatization of silicon-based nanoporous photonic crystals is presented as a method to immobilize peptides for the detection of protease enzymes in solution. A narrow-line-width rugate filter, a one-dimensional photonic crystal, is fabricated that exhibits a high-reflectivity optical resonance that is sensitive to small changes in the refractive index at the pore walls. To immobilize peptide in the pore of the photonic crystal, the hydrogen-terminated silicon surface was first modified with the alkene 10-succinimidyl undecenoate via hydrosilylation. The monolayer with the succinimide ester moiety at the distal end served the dual function of protecting the underlying silicon from oxidation as well as providing a surface suitable for subsequent derivatization with amines. The surface was further modified with 1-aminohexa(ethylene glycol) (EG(6)) to resist nonspecific adsorption of proteins common in complex biological samples. The distal hydroxyl of the EG(6) is activated using the solid-phase coupling reagent disuccinimidyl carbonate for selective immobilization of peptides as protease recognition elements. X-ray photoelectron spectroscopy analysis reveals high activation and coupling efficiency at each stage of the functionalization. Exposure of the peptide-modified crystals to the protease subtilisin in solution causes a change in the refractive index, resulting in a shift of the resonance to shorter wavelengths, indicating cleavage of organic material within the pores. The lowest detected concentration of enzyme was 37 nM (7.4 pmol in 200 microL).

Electrochemical DNA biosensors exploit the affinity of single-stranded DNA for complementary strands of DNA and are used in the detection of specific sequences of DNA with a view towards developing portable analytical devices. Great progress has been made in this field but there are still numerous challenges to overcome. This review for researchers new to the field describes the components of electrochemical DNA biosensors and the important issues in their design. Methods of transducing DNA binding events are discussed along with future directions for DNA biosensors.

The charge-transfer properties of DNA duplexes are exploited to produce a fast, simple, sensitive, and selective DNA biosensor by exposing the DNA recognition interface to a sample containing target DNA and the redox-active intercalator, anthraquinonemonosulfonic acid (AQMS). Electrochemistry from electron transfer through the DNA to AQMS intercalated into DNA duplexes can be differentiated from electrochemistry due to direct access of the AQMS to the electrode surface due to the difference in the environment of the AQMS giving a shift in the potential at which the molecule is reduced. The ability to distinguish between the two electrochemical signals enables DNA hybridization to be monitored in real time. This in situ detection scheme has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing either C-A or G-A single-base mismatches. The concentration detection limit of the biosensor is 0.5 nM (1 pmol) with an assay time of 1 h. The fact that the end user is only required to simultaneously add the sample containing the target DNA and AQMS gives a DNA biosensor that is highly compatible with PCR on chip technologies.

The efficient transfer of electrons between enzymes and electrodes is important for understanding the intrinsic redox properties as well as for developing protein-based biosensors and bioelectronic devices. One strategy to achieve efficient electron transfer to proteins is to build up the electrode inside the protein so that it is close to the redox-active center of the protein. To achieve this requires exceedingly small electrodes. Carbon nanotubes, which are as small as 1 nm in diameter, have the potential to be such electrodes. This chapter outlines recent research toward this goal via the self-assembly of vertically aligned single-walled carbon nanotubes on electrode surfaces followed by the subsequent attachment of proteins to the free ends of the tubes.

The label-free amperometric detection of a rabbit IgG antigen by an anti-rabbit IgG antibody is achieved by observing the electrochemistry at a glassy carbon electrode modified with antibody entrapped in an electrodeposited polypyrrole membrane. In a flow injection apparatus the electrode is pulsed between -0.2 and +0.4 V versus Ag/AgCl. The pulsing of the electrode switches the polypyrrole membrane between the oxidised and reduced states. When antigen is injected into the flow stream a change in current is observed at the electrode despite the antigen or antibody being redox inactive at the potentials employed. It is proposed that this current is due to a change in the flux of ions into and out of the polypyrrole matrix during a pulse when the poly-anionic antigen is present. The immunoreaction was reversible because the 200 ms pulse at each potential was too short to allow secondary bonding forces (hydrogen bonding and hydrophobic forces) which are responsible for the strength of the antibody-antigen complex to be established. The consequence of the reversibility of the antigen-antibody binding is a low apparent affinity constant but an easily regenerated recognition interface.

A label free electrochemical method of detecting DNA hybridisation is presented based on the change in flexibility between a single strand of DNA and a duplex causing an ion-gating effect where hybridisation opens up the electrode to access of ions.

A DNA hybridization biosensor based on long-range electron transfer that is capable of detecting DNA single-base mismatch is presented. A mixed self-assembled monolayer of single-stranded DNA (ss-DNA), thiolated at the 3' end, and 6-mercapto-1-hexanol was formed on a gold surface. This probe ss-DNA-modified gold surface was incubated in 2,6-disulfonic acid anthraquinone (AQDS) intercalator solution, rinsed, and placed in an AQDS-free buffer solution, whereupon voltammetric experiments were performed. No voltammetric peaks were observed for probe ss-DNA-modified gold electrodes. Upon DNA hybridization and incubation in AQDS, clear voltammetric peaks, consistent with the oxidation and reduction of AQDS, were observed. The absence of AQDS electrochemistry for ss-DNA-modified surfaces clearly shows the electrochemistry is due to long-range electron transfer through the DNA duplex. No peak currents were observed when the probe ss-DNA-modified surface was exposed to noncomplementary target DNA, but there was a diminution in current signal upon hybridization with C-A mismatched and a G-A mismatched targets.

An oxygen-rich fill-and-flow channel biosensor has been developed for the measurement of glucose in wine. Glucose oxidase (GOD), immobilised in carbon paste (CP), was located in a well adjacent to a downstream detector electrode. When the analyte solution flows, hydrogen peroxide produced in the enzyme reaction is swept down to the detector electrode. Mineral oil and Kel-F oil (poly(chlorotrifluorethylene)) were used to prepare an enzyme layer of GOD within a CP. The hydrophobicity of the CP confined the reaction between the enzyme and its substrate to the surface of the enzyme layer. The oxidation current of hydrogen peroxide was sensitive to the enzyme loading but insensitive to mass transport variations such as flow rate. This response was, therefore, limited by the kinetics of the reaction between the enzyme and the substrate. For Kel-F oil, which can support a high concentration of dissolved oxygen, good reproducibility and greater dynamic range was obtained and the response did not decrease after degassing for 40 min with argon. Analysis of wine samples showed good agreement with the values obtained by spectrophotometric enzyme assay.