Biosensor Array Research Articles

Capacitive Sensor Estimation Based on Self-Configurable Reference Capacitance

A number of capacitance readout systems presented in the literature and covered by recent patents are briefly described while a biosensor array readout system developed by the authors is presented in this paper. This biosensor array readout system is based on a charge sensitive amplifier that estimates accurately the difference between a selfconfigurable reference capacitance and a biosensor. In the specific implementation, the self-configurable reference capacitance is automatically configured to a value between 0 and 630pF in steps of 10pF. The configuration of the reference capacitance is controlled by a simple finite state machine that can be implemented using a simple CPLD (Complex Programmable Logic Device) such as Altera EPM7064SLC44-10. The proposed system can be used to measure an absolute biosensor capacitance of up to 640pF with a theoretical sensitivity of 2.5fF. The 2.5fF resolution can be achieved using a low cost/high accuracy 12-bit ADC (Analogue/Digital Converter) like a one that has been described by the authors in a recent patent instead of a 17-bit ADC that would be required to measure the biosensor capacitance value with the same accuracy if a constant 320pF reference capacitor had been used. For this reason, the proposed architecture leads to a lower area/power and higher accuracy implementation compared to a read out circuit that would use a fixed reference capacitance. Although the design of the proposed system was inspired by the development of a biosensor array readout system, a similar architecture can be used by any sensor application that should support a broad capacitive input range with high resolution. Keywords: Biosensor readout, configurable reference capacitors, analogue-to-digital conversion, charge sensitive amplifier, switched capacitance interface.

Carbamate Insecticide Sensing Based on Acetylcholinesterase/Prussian Blue-Multi-Walled Carbon Nanotubes/Screen-Printed Electrodes

Prussian blue (PB)-multi-walled carbon nanotubes (MWCNTs) modified screen-printed electrodes (SPEs) were used to immobilize enzyme acetylcholinesterase (AChE) for carbamate insecticide sensing. The synthesized hybrid PB-MWCNTs had high stability at pH values in the range of 5–10 and presented a porous and homogeneous microenvironment to entrap enzyme molecules. The generated hybrids not only acted as carriers of acetyl cholinesterase, but also promoted electron-transfer reactions because of the synergistic effects between MWCNTs and PB. Under the optimal conditions, the response of the sensor was proportional to acetylthiocholine (ATCh) concentrations ranging from 0.1 mM to 0.6 mM, with a sensitivity of 21.97 µA · mM−1 · cm−2. The sensors were further used to detect pesticides, and the inhibition rate of pirimicarb was proportional to the logarithm of its concentration ranging from 1.0 × 10−6 to 1.0 g · L−1, with a limit of detection (LOD) equal to 5.32 × 10−8 g · L−1. In order to evaluate the performance of the detection system, the sensors were applied to determine pirimicarb in water samples and exhibited high sensitivity and good stability. The detection system is fast, simple for analysis of pirimicarb in environmental samples, and could provide a semi-automated analytical system through further improvement in biosensor arrays.

Colorimetric surface plasmon resonance imaging (SPRI) biosensor array based on polarization orientation

A colorimetric surface plasmon resonance (SPR) imaging biosensor array based on polarization orientation rotation is presented in this paper. It measures the spectral characteristic variations caused by the steep phase difference between the p- and s-polarization occurring at surface plasmon excitation. It provides one-order of magnitude sensor resolution improvement comparing to existing phase-sensitive SPR imaging sensors and the two-dimensional (2D) sensing capability of the imaging sensor enables multiplex, high throughput array based simultaneous detection for a range of different bio-molecular interactions. Experiments on the binding interactions detection between anti-bovine serum albumin (anti-BSA) and BSA antigen have been performed. All binding interactions occurred at 5×4 protein array were real-time monitored simultaneously. A sensor resolution of 8.26ng/ml (125pM) has been demonstrated, which is one-order of magnitude (12 times) better than the detection limit reported by existing phase-sensitive SPR imaging sensors in the literature, while no time-consuming phase modulation and phase extraction processes are required. Furthermore, the optical colorimetric image read-out of the sensor is easy to be identified by the end users comparing to conventional intensity or phase information. The colorimetric SPR imaging biosensor array can find promising potential applications in high throughput clinical disease diagnosis, protein biomarkers screening and drug screening.

Biosensor array based on the in situ detection of quantum dots as electrochemical label

A simple, fast, and inexpensive miniaturized electrochemical biosensor array with in situ detection of CdSe/ZnS quantum dots (QDs) is presented.This biosensor uses the affinity reaction between streptavidin and biotin, for biotin determination, but the methodology could be extended to other clinical biomarkers. Individual screen-printed carbon electrodes (SPCEs) and 8-channel screen-printed carbon electrochemical arrays (8xSPCEs) were modified with streptavidin and its analytical performance was evaluated using QDs labeled biotin (biotin-QD). After the biological reaction, Cd2+ ions released from the dissolution of the nanoparticles were determined in situ by voltammetric stripping. A linear range of 5×10−10–5×10−9M and a limit of detection of 2×10−10M (in terms of QD) were achieved for SPCEs (5.0% RSD, n=3). For 8xSPCEs, a linear range of 1×10−9–1.2×10−8M and a limit of detection of 3×10−10M (in terms of QD) were obtained (8.5% RSD, n=3). A one-step competitive bioassay between biotin and biotin-QD was carried out using the biosensor array. A linear range of two orders of magnitude (1×10−9–1×10−7M) and a limit of detection of 1.4×10−9M were achieved for biotin (6.4% RSD, n=3).

Microbial fuel cell type biosensor for specific volatile fatty acids using acclimated bacterial communities

Volatile fatty acid (VFA) concentration is one of the most important parameters for monitoring bio-processes such as anaerobic digestion and microbial fuel cells. In this study the correlation between VFA concentration and current/voltage responses and electrochemical properties by using the MFC technology was evaluated. The discrimination between different species of VFA by using two methods i.e., coulombic efficiency and cyclic voltammetry was investigated. Columbic efficiency gave a slow response of greater than 20h, particularly at concentration levels of 20mgl−1. By using cyclic voltammetry to measure the oxidation peak at a consistent scan rate showed linear correlation to VFA concentration and peak current produced, up to <40mgl−1) in a rapid response time of 1–2min. The results presented showed good correlations between the individual VFA species concentration and charge, and also current generated. A MFC based biosensor array was produced capable of measuring individual acetate, propionate and butyrate concentrations with sensitivity down to 5mgl−1 and up to 40mgl−1.

A microfluidic paper-based electrochemical biosensor array for multiplexed detection of metabolic biomarkers

Paper-based microfluidic devices have emerged as simple yet powerful platforms for performing low-cost analytical tests. This paper reports a microfluidic paper-based electrochemical biosensor array for multiplexed detection of physiologically relevant metabolic biomarkers. Different from existing paper-based electrochemical devices, our device includes an array of eight electrochemical sensors and utilizes a handheld custom-made electrochemical reader (potentiostat) for signal readout. The biosensor array can detect several analytes in a sample solution and produce multiple measurements for each analyte from a single run. Using the device, we demonstrate simultaneous detection of glucose, lactate and uric acid in urine, with analytical performance comparable to that of the existing commercial and paper-based platforms. The paper-based biosensor array and its electrochemical reader will enable the acquisition of high-density, statistically meaningful diagnostic information at the point of care in a rapid and cost-efficient way.

High density CMOS electrode array for high-throughput and automated cell counting

A high-density electrical-impedance spectroscopy (EIS) biosensor array has been developed for high-throughput and automated counting of specific breast tumor MCF-7 cells. The biosensor array (96×96) consists of densely packed electrodes. Each of the square electrodes has an edge-length of 22μm to capture single MCF-7 cell. The electrodes are electrically addressable by decoding circuit built underneath by 0.18μm CMOS process. EIS spectra of electrodes were recorded over a wide frequency range with and without the presence of cell. Data were numerically fitted with the equivalent circuit model to extract important sensing parameters including cell impedance, electrode impedance, sealing resistance and spreading resistance. Results revealed a distinctive impedance increase from 50kHz to 500kHz, where the maximum (∼21% increment) occurs at 200kHz. Enumeration of circulating tumor cells (CTCs) was performed by scanning an interrogation area of hundred electrodes at the optimum frequency. The developed EIS platform has demonstrated the electrical impedance detection with single cell resolution and enumeration with mapping accuracy of ∼90%.

Bioelectronic tongue and multivariate analysis: A next step in BOD measurements

Seven biosensors based on different semi-specific and universal microorganisms were constructed for biochemical oxygen demand (BOD) measurements in various synthetic industrial wastewaters. All biosensors were calibrated using OECD synthetic wastewater and the resulting calibration curves were used in the calculations of the sensor-BOD values for all biosensors. In addition, the output signals of all biosensors were analyzed as a bioelectronic tongue and comprehensive multivariate data analysis was applied to extract qualitative and quantitative information from the samples. In the case of individual biosensor measurements, most accurate result was gained when semi-specific biosensor was applied to analyze sample specific to that biosensor. Universal biosensors or biosensors semi-specific to other samples underestimated the BOD7 of the sample 10–25%. PLS regression method was used for the multivariate calibration of the biosensor array. The calculated sensor-BOD values differed from BOD7 less than 5.6% in all types of samples. By applying PCA and using three first principal components, giving 99.66% of variation, it was possible to differentiate samples by their compositions.

Piezoelectric olfactory receptor biosensor prepared by aptamer-assisted immobilization

Inspired by the very high sensitivity and specificity of biological olfactory system, engineers pay much attention to biomimetic olfactory biosensors due to their excellent performance and great commercial prospects. In this study we presented an olfactory receptor (OR)-based piezoelectric biosensor with high sensitivity and specificity for the odorant detection, in which aptamers were employed for the specific and effective olfactory receptor immobilization to improve the overall performance of biosensors. An olfactory receptor of C. elegances, ODR-10, was expressed heterologously in HEK-293cells with a hexahistidine (His6) tag on the N terminus. His6-tagged ODR-10 was extracted from the plasma membrane of transfected HEK-293cells and used as the sensing element of biosensors. Anti-His6 aptamers were immobilized covalently onto the gold surface of piezoelectric quartz crystal microbalance (QCM) electrode to capture the His6-tagged ODR-10 specifically, as well as achieve the purification of ODR-10 simultaneously. The immobilization procedures were characterized by electrochemical methods and scanning probe microscopy (SPM). Additionally, physical adsorption method was used as a control method to illustrate the influences of immobilization methods on the performances of OR-based biosensors. The results demonstrated that the piezoelectric biosensor prepared by aptamer-assisted immobilization method showed high specificity and improved sensitivity. The detection limit was as low as 1.5ppm (v/v). Additionally, the aptamer-assisted receptor immobilization method has great potential to become a universal protein immobilization technique in biosensing, and be applied into the development of OR-based biosensor array for the simultaneous detection of several odorants. This aptamer-assisted olfactory receptor biosensor has great application prospects in many fields, such as food safety, environmental monitoring, and disease diagnosis.

Detection and discrimination of alpha-fetoprotein with a label-free electrochemical impedance spectroscopy biosensor array based on lectin functionalized carbon nanotubes

A label-free electrochemical impedance spectroscopy (EIS) biosensor for the sensitive determination and discrimination of alpha-fetoprotein (AFP) was developed by employing wheat-germ agglutinin (WGA) lectin as molecular recognition element. The EIS biosensor was fabricated by adsorbing carboxyl-functionalized single-wall carbon nanotubes (SWNTs) onto a screen-printed carbon electrode (SPCE) and subsequently covalently coupling WGA onto the surface of the SWNTs-modified electrode. Upon binding of AFP to the biosensor, the electron transfer resistance was increased and the increase in the electron transfer resistance was linearly proportional to the logarithm of the concentration of AFP in the range from 1 to 100ng/L with a detection limit of 0.1ng/L. It was found that the employment of SWNTs as immobilization platform could reduce the background and enhance the EIS response. Moreover, the lectin-based biosensor array fabricated with different lectins was used to evaluate the glycan expression of AFP N-glycan and discriminate AFP between healthy and cancer patients serum samples. This work demonstrates that the employment of carbon nanotubes as immobilization platform and lectin as molecular recognition element in biosensor array is a promising approach for the determination and discrimination of glycoproteins for cancer diagnosis. The strategy proposed in this work could further be used for high-throughput, label-free profiling of the glycan expression of cancer-related glycoproteins and to develop methods for cancer diagnosis in the early stages.

A CMOS magnetic microbead-based capacitive biosensor array with on-chip electromagnetic manipulation

Magnetic microbeads are widely used in biotechnology and biomedical research for manipulation and detection of cells and biomolecules. Most lab-on-chip systems capable of performing manipulation and detection require external instruments to perform one of the functions, leading to increased size and cost. This work aims at developing an integrated platform to perform these two functions by implementing electromagnetic microcoils and capacitive biosensors on a CMOS (complementary metal oxide semiconductor) chip. Compared to most magnetic-type sensors, our detection method requires no externally applied magnetic fields and the associated fabrication is less complicated. In our experiment, microbeads coated with streptavidin were driven to the sensors in the center of microcoils with functionalized anti-streptavidin antibody. Detection of a single microbead was successfully demonstrated using a capacitance-to-frequency readout. The average capacitance changes for the experimental and control groups were −5.3fF and −0.2fF, respectively.

ZnO nanowire array-templated LbL self-assembled polyelectrolyte nanotube arrays and application for charged drug delivery

Vertically oriented and robust polyelectrolyte nanotube arrays with high density, large area and high uniformity were successfully grown on substrates by a ZnO nanowire array-templated layer-by-layer (LbL) self-assembly approach for the first time, and were further used to deliver charged drugs, showing that they not only possess pH-responsive loading property, but also significantly enhance the loading capacity and sustained release time. This work could be extended to fabricate polyelectrolyte nanotube arrays with different polyelectrolyte combinations, including weak polyelectrolyte/weak polyelectrolyte, weak polyelectrolyte/strong polyelectrolyte and strong polyelectrolyte/strong polyelectrolyte. With the great versatility to use various substrates and building blocks, the polyelectrolyte nanotube arrays may have great potential for broad applications such as biosensor arrays, bioreactor arrays and optoelectronics.

Metal enhanced fluorescent biosensing assays for DNA through the coupling of silver nanoparticles

A novel biosensor array for DNA detection based on the coupling of silver nanoparticles is developed. Two kinds of DNA–silver nanoparticle conjugates were immobilized on the aldehyde modified slides. The fluorescence intensity of Cy3 within the proximity of the conjugates was enhanced due to the coupling of the silver nanoparticles based on hybridization of target DNA and the conjugates. The fluorescence intensity increases with the concentration of target DNA, and the enhancement factor is about 2.4 at the optimized conditions. The results gave a good linear correlation between DNA concentration and fluorescence intensity at DNA concentration range from 12.8 pM to 40 nM. The biosensors constructed on the slide as a microarray offer a unique approach for DNA detection with the advantages of high sensitivity and rapidity. The fluorescence enhancement was attributed to the coupling between the silver nanoparticles. This new strategy opens the possibility for the preparation of highly enhanced plasmonic substrate for DNA analysis.

Analysis of copper nanoparticles toxicity based on a stress-responsive bacterial biosensor array

The rapid development in nanoparticle production and application during the past decade requires an easy, rapid, and predictive screening method for nanoparticles toxicity assay. In this study, the toxicological effects and the source of toxicity of copper nanoparticles (CuNPs) are investigated based on a stress-responsive bacterial biosensor array. According to the responses of the biosensing strains, it is found that CuNPs induce not only oxidative stress in E. coli, but also protein damage, DNA damage, and cell membrane damage, and ultimately cause cell growth inhibition. Through enzyme detoxification analysis, the toxicological effects of CuNPs are traced to H(2)O(2) generation from CuNPs. Rapid copper release from CuNPs and Cu(I) production are observed. The oxidation of the released Cu(I) has a close relation to H(2)O(2) production, as tris-(hydroxypropyltriazolylmethyl) amine, the specific Cu(I) chelator, can largely protect the cells from the toxicity of CuNPs. In addition, the TEM study shows that CuNPs can be adsorbed and incepted fast by the cells. Comparatively, copper microparticles are relatively stable in the system and practically non-toxic, which indicates the importance of toxic estimation of materials at the nanoscale. In addition, the Cu(II) ion can induce protein damage, membrane damage, and slight DNA damage only at a relatively high concentration. The current study reveals the preliminary mechanism of toxicity of CuNPs, and suggests that the stress-responsive bacterial biosensor array can be used as a simple and promising tool for rapid screening in vitro toxicity of nanoparticles and studying the primary mechanism of the toxicity.

Direct growth of nanotubes and graphene nanoflowers on electrochemical platinum electrodes

Multi-walled carbon nanotubes and graphene nanoflowers were grown by a catalytic chemical vapor deposition process on metal surfaces. Electrodeposition was used as a versatile technique to obtain three different iron catalyst coatings on platinum microelectrodes. The influence of growth parameters on carbon deposits was investigated. Characterization was carried out by scanning electron microscopy and Raman spectroscopy. A chemical treatment in sulphuric acid produced an increased voltammetric background current. In Raman spectra, the effect of the chemical treatment is seen as a more pronounced sp(3) hybridisation mode of C resulting from surface functionalization of the C nanomaterials. Overall, the hybrid electrodes we produced exhibit a promising performance for oxidase-based array biosensors. Therefore, our study opens the possibility of integrating the hybrid electrodes in biochip applications.

Arrays of nanoelectromechanical biosensors functionalized by microcontact printing

The biofunctionalization of nanoelectromechanical systems (NEMS) is critical for the development of new classes of biosensors displaying improved performance and higher levels of integration. In this paper we propose a modified microcontact process (μCP) in order to biofunctionalize arrays of NEMS with a probe molecule on the active sensing areas together with an anti-fouling layer on the passive areas in a single, self-aligned step. We demonstrate the adequate functionalization/anti-fouling of arrays of freestanding nanocantilevers as dense as 105 nanostructures cm−2 by using both fluorescence microscopy and dynamic measurements of the structures’ resonant frequency. The proper bioactivity of an antibody deposited onto the cantilevers and the blocking property of a bovine serum albumin layer are both assessed by incubating specific and non-specific tagged secondary antibodies followed by fluorescence imaging. Furthermore, measurement of the resonant frequency of the nanocantilevers before and after functionalization and biological recognition demonstrate that using μCP for device functionalization does not damage the nanostructures and preserves the mechanical sensing capability of our NEMS.

Parallel recording of neurotransmitters release from chromaffin cells using a 10×10 CMOS IC potentiostat array with on-chip working electrodes

Neurotransmitter release is modulated by many drugs and molecular manipulations. We present an active CMOS-based electrochemical biosensor array with high throughput capability (100 electrodes) for on-chip amperometric measurement of neurotransmitter release. The high-throughput of the biosensor array will accelerate the data collection needed to determine statistical significance of changes produced under varying conditions, from several weeks to a few hours. The biosensor is designed and fabricated using a combination of CMOS integrated circuit (IC) technology and a photolithography process to incorporate platinum working electrodes on-chip. We demonstrate the operation of an electrode array with integrated high-gain potentiostats and output time-division multiplexing with minimum dead time for readout. The on-chip working electrodes are patterned by conformal deposition of Pt and lift-off photolithography. The conformal deposition method protects the underlying electronic circuits from contact with the electrolyte that covers the electrode array during measurement. The biosensor was validated by simultaneous measurement of amperometric currents from 100 electrodes in response to dopamine injection, which revealed the time course of dopamine diffusion along the surface of the biosensor array. The biosensor simultaneously recorded neurotransmitter release successfully from multiple individual living chromaffin cells. The biosensor was capable of resolving small and fast amperometric spikes reporting release from individual vesicle secretions. We anticipate that this device will accelerate the characterization of the modulation of neurotransmitter secretion from neuronal and endocrine cells by pharmacological and molecular manipulations of the cells.

Towards an integrated biosensor array for simultaneous and rapid multi-analysis of endocrine disrupting chemicals

In this paper we propose the construction and application of a portable multi-purpose biosensor array for the simultaneous detection of a wide range of endocrine disruptor chemicals (EDCs), based on the recognition operated by various enzymes and microorganisms. The developed biosensor combines both electrochemical and optical transduction systems, in order to increase the number of chemical species which can be monitored. Considering to the maximum residue level (MRL) of contaminants established by the European Commission, the biosensor system was able to detect most of the chemicals analysed with very high sensitivity. In particular, atrazine and diuron were detected with a limit of detection of 0.5nM, with an RSD% less than 5%; paraoxon and chlorpyrifos were revealed with a detection of 5μM and 4.5μM, respectively, with an RSD% less than 6%; catechol and bisphenol A were identified with a limit of detection of 1μM and 35μM respectively, with an RSD% less than 5%.

Detection of odorant molecules via surface acoustic wave biosensor array based on odorant-binding proteins

In this paper, we present an array of biosensors for vapour phase detection of odorant molecules based on surface acoustic wave (SAW) resonators coated with odorant-binding proteins (OBPs). For the first time, the sensing capabilities of three different OBPs, as sensitive layers for SAW devices, are studied and compared. The SAW biosensor array is composed of three SAW devices coated by the droplet method with the wild-type OBP from cow (wtbOBP), a double mutant of the OBP from cow (dmbOBP) and the wild-type OBP from pig (wtpOBP). An uncoated device is used to compensate the variations of the environmental parameters. The SAW devices consist of two-port resonators fabricated on quartz (ST-cut, x propagation) with electrodes made of aluminium covered with a thin gold film (2nm thick). The obtained surface densities of OBP layers are between 1.18×10−6kg/m2 and 2.31×10−6kg/m2 and were calculated measuring the resonant frequency shift of the SAW devices after the coating. The SAW biosensor array was tested in nitrogen upon exposure to vapours of R-(–)-1-octen-3-ol (octenol), in the range of concentration between 13 and 61ppm, and R-(–)-carvone (carvone), in the range between 9 and 80ppm. The highest sensitivity for detection of octenol (25.9Hz/ppm) was obtained using the wtpOBP-based SAW biosensor, while the highest sensitivity for detection of carvone (9.2Hz/ppm) was obtained using the dmbOBP-based SAW biosensor.

Mussel adhesive protein-based whole cell array biosensor for detection of organophosphorus compounds

A whole cell array biosensor for the efficient detection of neurotoxic organophosphate compounds (OPs) was developed through the immobilization of recombinant Escherichia coli cells containing periplasmic-expressing organophosphorus hydrolase (OPH) onto the surface of a 96-well microplate using mussel adhesive protein (MAP) as a microbial cell-immobilizing linker. Both the paraoxon-hydrolyzing activity and fluorescence microscopy analyses demonstrated that the use of MAP in a whole cell biosensor increased the cell-immobilizing efficiency and enhanced the stability of immobilized cells compared to a simple physical adsorption-based whole cell system. Scanning electron microscopic analyses also showed that the E. coli cells were effectively immobilized on the MAP-coated surface without any pretreatment steps. The whole cell array biosensor system, prepared using optimal MAP coating (50μg/cm2) and cell loading (4OD600), detected paraoxon levels as low as 5μM with high reproducibility, and its quantitative detection range was ∼5–320μM. The MAP-based whole cell array biosensor showed a good long-term stability for 28 day with 80% retained activity and a reusability of up to 20 times. In addition, paraoxon in tap water was also successfully detected without a reduction in sensitivity. Our results indicate that the proposed MAP-based whole cell array system could be used as a potential platform for a stable and reusable whole cell biosensor.