Ultrasensitive immunosensing of tuberculosis CFP-10 based on SPR spectroscopy

An antigen (Ag), CFP-10, found in tissue fluids of tuberculosis (TB) patients may be an ultimate candidate for use as a sensitive TB marker with a sensing method for early simplified diagnosis of TB. In this study, chemical and optical optimizations were carried out using novel immuno-materials for establishment of a self-assembled surface plasmon resonance (SPR) optical immunosensor system for detection of CFP-10, which is valuable for pre-clinical work, prior to conduct of massive clinical observations. For creation of a simple sensing interface, a monoclonal antibody (anti-CFP-10) was immobilized directly on a gold surface, followed by blocking with cystamine. Orientation and accessibility of anti-CFP-10 were assessed by the selective binding of CFP-10. Recent results indicate that the reusability of the sensor chip adopting the cystamine method was found to be preferable to other immobilization methods. A linear relationship was well correlated between SPR angle shift and CFP concentrations in the range from 100 ng mL⁻¹ to 1 μg mL⁻¹. Modification of the SPR chip with antibody provides a simple experimental platform for investigation of isolated proteins under experimental conditions resembling those of their native environment.     

基于SPR干涉成像传感法检测大蒜素刺激胃癌细胞的响应

针对细胞响应传感的要求,提出了SPR干涉成像传感细胞响应的方法,设计制作新颖的微流体池,构建用于活细胞水平分子传感和检测的系统。在此基础上,先检测和分析细胞在微流体池中的培养状态,再检测大蒜素对人胃癌细胞系BGC823刺激响应。实验结果表明,胃癌细胞贴附生长良好,微流体池可提供细胞生存的微环境;6 h后大蒜素刺激和对照的SPR信号响应值分别为0.15 RIRF和0.35 RIRF,大蒜素对胃癌细胞系BGC823细胞具有明显抑制作用。在持续提高精度、可靠性以及丰富有关工艺技术后,有可能建立完整的药物刺激细胞响应检测技术平台,为药物发现和临床化疗方案优化做出贡献。     

Tethered DNA scaffolds on optical sensor platforms for detection of hipO gene from Campylobacter jejuni

DNA biosensors have gained increased attention over traditional diagnostic methods due to their fast and responsive operation and cost-effective design. The specificity of DNA biosensors relies on single-stranded oligonucleotide probes immobilized to a transduction platform. Here, we report the development of biosensors to detect the hippuricase gene (hipO) from Campylobacter jejuni using direct covalent coupling of thiol- and biotin-labeled single-stranded DNA (ssDNA) on both surface plasmon resonance (SPR) and diffraction optics technology (DOT, dotLab) transduction platforms. This is the first known report of the dotLab to detect targeted DNA. Application of 6-mercapto-1-hexanol as a spacer thiol for SPR gold surface created a self-assembled monolayer that removed unbound ssDNA and minimized non-specific detection. The detection limit of SPR sensors was shown to be 2.5 nM DNA while dotLab sensors demonstrated a slightly decreased detection limit of 5.0 nM (0.005 μM). It was possible to reuse the SPR sensor due to the negligible changes in sensor sensitivity (∼9.7 × 10⁻⁷ ΔRU) and minimal damage to immobilized probes following use, whereas dotLab sensors could not be reused. Results indicated feasibility of optical biosensors for rapid and sensitive detection of the hipO gene of Campylobacter jejuni using specific ssDNA as a probe.     

Highly accurate and sensitive surface plasmon resonance sensor based on channel photonic crystal waveguides

A highly sensitive surface plasmon resonance (SPR) sensor based on channel photonic crystal waveguide (PCW) is proposed. The PCW is based on widely used lithographic and nano-fabrication compatible materials like TiO₂ and SiO₂. Gold has been used as a SPR active metal. By rigorously optimizing the different waveguide parameters, we have shown that there is significant transfer of modal power around phase-matching or resonance wavelength which has been utilized to design a compact and highly sensitive sensor for lab on chip. The ultra narrow width (∼765 pm for an interaction length of 10 mm) of surface plasmon resonance curve and sensitivity as high as 7500 nm-RIU⁻¹ will open a new window for bio-chemical sensing applications.     

A polymer lab chip sensor with microfabricated planar silver electrode for continuous and on-site heavy metal measurement

This paper presents a reusable polymer lab chip sensor for continuous and on-site heavy metal monitoring in nature. In particular, detection of lead (Pb(II)), which is the most common heavy metal pollutant, has been performed using the proposed lab chip sensor. The miniaturized lab chip sensor consists of a microfabricated silver working electrode that replaces the conventional mercury and bismuth electrodes, an integrated silver counter and quasi-reference electrode, and microfluidic channels. The proposed sensor targets on-site environmental monitoring in a continuous fashion without disturbing or contaminating the sensing environment when it is reused. The reusability of the miniaturized lab chip sensor was characterized through forty-three consecutive measurements in non-deoxygenating standard solutions inside the microchannels using square-wave anodic stripping voltammetry (SWASV). With only 13.5 μL of sample volume the sensor chip showed a correlation coefficient of 0.998 for the Pb(II) concentration range of 1-1000 ppb with the limit of detection of 0.55 ppb at 300 s deposition time. The peak potentials during the forty-three consecutive SWASV measurements showed a relative standard deviation of 1.0%, with a standard deviation of 0.005 V. The high repeatability and linearity of the sensor over the large, three orders of magnitude, dynamic range of 1-1000 ppb showed that the developed sensor chip can be reused for a variety of on-site measurements such as for soil pore water or groundwater, using only micro-volumes.     

Study on design and application of fully automatic miniature surface plasmon resonance concentration analyzer

A fully automatic miniature surface plasmon resonance (SPR) concentration analyzer having high performance and low cost and developed using a Spreeta™ sensor was designed for field applications and concentration analysis. As in the case of Biacore™ instruments, the automatic sampling system of this device can introduce air segments between the sample/regeneration solution and buffer solution in the pipeline, which effectively prevents mixing of the solutions. A temperature sensor (AD 590) and temperature compensation method are used, which make the device insensitive to temperature fluctuations. A real-time data-smoothing algorithm for the SPR detection data is adopted; this can reduce the noise level to 5 × 10⁻⁷ RIU (refractive index units). The noise level of the sensorgram is 3.5% of the original level. Two types of self-prepared sensing chips—SMX-BSA (bovine serum albumin coated with sulfamethoxazole) and SMX-CM5 (carboxymethyl dextran coated with sulfamethoxazole)—are used to analyze the concentrations of sulfamethoxazole (SMX) standard solutions. Each chip's SMX calibration curve is established within the measurement range of 0-2000 ng/ml, and both limits of detection (LOD) are 2 ng/ml. One cycle of assay time is less than 15 min.     

Micro-piezoelectric immunoassay chip for simultaneous detection of Hepatitis B virus and α-fetoprotein

In this paper, a piezoelectric diaphragm-based immunoassay chip was developed to simultaneously detect anti-Hepatitis B virus (HBV) and anti-alpha-fetoprotein (AFP). The chip was fabricated by micro-machining technology and consists of eight individual circular sensors with a diameter of 800 μm. Hepatitis B surface antigen (HBsAg), Hepatitis C core antigen (HBcAg) and AFP as the probe molecules were immobilized on different sensing spots on the chip. A solution containing anti-HBsAg and anti-AFP was applied into the reaction chambers in all sensors of the chip, and significant frequency shifts were only observed in the sensors with HBsAg and AFP for immunoassay detection. The fluorescence image further confirmed the successful detection of anti-HBsAg and anti-AFP. The total assay time was less than 2 h. The frequency shift-based calibration curves show a detection limit of 0.1 ng/ml and a dynamic detection range of 0.1-10,000 ng/ml for both anti-HBsAg and anti-AFP, respectively, thus demonstrating that the developed piezoelectric immunoassay chip has potential applications for rapid, specific, sensitive, and multiple detections of HBV.     

A multichannel surface plasmon resonance sensor using a new spectral readout system without moving optics

Surface plasmon resonance (SPR) sensors with spectral interrogation provide a high refractive index resolution, a large dynamic range and a fixed optical detection module. In this work, we propose a new multichannel spectral detection unit that uses only one spectrometer to measure the reflection spectrum from multiple sensing spots serially without any mechanical movement. This spectral detection unit is designed based on a spatial light modulator (SLM) configured as a programmable optical aperture for the spectrometer. To demonstrate this concept, a five-channel laboratory SPR prototype was built based on the proposed multichannel detection unit, and we evaluated the device's sensitivity and resolution using a refractive index test. Refractive index resolution of 1.4 × 10⁻⁶ refractive index units (RIU) can be reached using the five-channel prototype. This sensor is suitable for low-cost multichannel biosensing applications that do not contain fast kinetics.     

Rapid detection of bacterial cell from whole blood: Integration of DNA sample preparation into single micro-PCR chip

A novel bacterial cell detection method from blood samples has been developed for molecular diagnostics. Functional integration of DNA sample preparation into polymerase chain reaction (PCR) chip enabled detection of pathogenic bacterial cells in a single microchip. Surface-modified micropillars possessing affinity for bacterial cells were fabricated inside a PCR chip, and reaction conditions were optimized to render the microchip with high surface-to-volume ratio PCR-compatible. After bacterial cells were captured on the micropillars from whole blood and PCR inhibitors were washed out, PCR mixture was injected to allow real-time amplification of DNA extracted from the isolated cells. Cell enrichment effect produced by volume reduction from large initial sample to small micro-PCR chip chamber led to increased detection sensitivity. Moreover, the developed method from sample preparation to detection of bacterial cells from whole blood took less than 1 h. These results demonstrated that the surface-modified pillar-packed microchip would be a practical approach for integration into Lab-On-a-Chip (LOC) to enable point-of-care genetic analysis.     

Determination of 3-nitrotyrosine in human urine samples by surface plasmon resonance immunoassay

This paper describes the detection of a low-molecular weight molecule, 3-nitrotyrosine (3-NT) (∼226 Da), in human urine by coupling indirect inhibition assay with a surface plasmon resonance (SPR) sensor. 3-NT antibody (anti-3-NT Ab, mouse IgG) was used in this assay. An optimal antibody concentration has been measured at 27.9 μg/mL in order to obtain the best performance of the sensor surface. The lowest detection limit for 3-NT with this method is 4.7 ng/mL (S/N = 3). Sensor reliability was demonstrated by good specificity, intra-assay and inter-assay relative standard deviations <8%, average recovery of 107.68 ± 19.4% and sensor surface (self-assembled monolayer) stability through more than 200 regeneration cycles and 15 days of repeated measurement. This is the first SPR biosensor assay of 3-NT in human urine. The high stability of the SPR sensor surface underlies the potential of the SPR method as a low cost diagnostic tool for clinical detection of 3-NT.     

Integration of thin film amorphous silicon photodetector with lab-on-chip for monitoring protein fluorescence in solution and in live microbial cells

Green fluorescent protein (GFP) and other fluorescent protein variants are widely used as powerful tools to tag and monitor complex biological processes at the cellular, tissue, and organism level. A micrometer-scale GFP detection system is demonstrated in which a p-i-n thin-film amorphous silicon light sensitive layer microfabricated on a glass substrate is integrated with an amorphous silicon carbon alloy absorption filter and an ultra-thin PDMS sheet to detect intracellular expression of GFP. GFP fluorescence was detected in aqueous solution in the nM range, and inside living cells (Escherichia coli) expressing GFP in the 10⁶ cell/mL range. The temporal evolution of the integrated fluorescence from promoter-induced GFP production was monitored in E. coli in solution over 12 h using the detection system. This microfabricated thin-film detection system can potentially be developed into an array-based sensing of GFP allowing for high-throughput and multiplexed analysis of biological and biomedical samples.     

ZeptoFarad capacitance detection with a miniaturized CMOS current front-end for nanoscale sensors

The experimental detection of capacitance variations with a resolution as low as few zeptoFarads (10⁻²¹ F) is presented. This is achieved by means of a CMOS ultra-low-noise and wide-bandwidth current sensing circuit, coupled to a lock-in amplifier to perform capacitance and conductance measurements in a frequency range from DC to 1 MHz. The adoption of an integrated implementation, based on an original circuital topology, provides miniaturization and performance improvement. The mm-sized chip can be easily integrated in extremely compact sensing setups. Resolution limits are analyzed in detail and experimentally investigated by means of a mechanical fixture that converts micrometric linear displacement into sub-aF capacitance steps. The experimental results match the theoretical expectation down to a resolution of 5 zF_rms (6 V at 100 kHz, with a 100 ms time constant). The achieved current resolution of 15 fA_rms (at ∼ms time scale) and the tracking of 40 zF capacitance steps demonstrate how the proposed read-out circuit can serve as a versatile tool for the development of nanosensors.     

Highly sensitive label-free immunosensor for ochratoxin A based on functionalized magnetic nanoparticles and EIS/SPR detection

A label-free immunosensor for the detection of ochratoxin A (OTA) based on use of magnetic nanoparticles (MNPs) was developed. A gold electrode was modified using bovine serum albumin conjugate with a glutaraldehyde-thiolamine linker, creating a layer that prevents non-specific binding of OTA on gold. The OTA antibodies were attached to MNPs using the carbodiimide chemistry and afterwards were immobilized on the modified gold electrode using a strong magnetic field. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) were used to characterize each step in immunosensor development. The impedance variation due to the specific antibody-OTA interaction was correlated with the OTA concentration in the samples. The increase in electron-transfer resistance values was proportional to the concentration of OTA on a linear range between 0.01 and 5 ng/mL, with a detection limit of 0.01 ng/mL. SPR measurements showed a larger response range (1-50 ng/mL) with a detection limit of 0.94 ng/mL. Analytical results were in accordance with standard ELISA test kit.     

On-chip glucose biosensor based on enzyme entrapment with pre-reaction to lower interference in a flow injection system

In this study, poly(3,4-ethylenedioxythiophene), PEDOT, was electropolymerized on a sensing chip to entrap glucose oxidase (GOD). The interdigitated array (IDA) electrode and microfluidic channel of the sensing chip was fabricated by photolithography. The IDA electrodes consist of two working electrodes in which one (WE1) was the enzyme-modified electrode and the other was a Pt (WE2) for eliminating noise effect. The microfluidic channel was formed on etched silicon by PDMS (poly(dimethylsiloxane)). In the flow injection analysis, a 0.7 V (vs. Ag/AgCl) was set on enzyme electrode to detect the catalytic product, H₂O₂, and the sensing signal was calibrated using the passed charge rather than the peak current. The linear relationship between the charges and the glucose concentrations, ranging from 1 to 10 mM, was obtained with a sensitivity of 157 μC cm⁻² mM⁻¹. Besides, the response time and the recovery time were about 15 and 35-75 s, respectively. In real human sample test, the error of single-potential and bi-potential were about 140% and 13%, respectively, comparing to the standard value, indicating that the WE2 can lower the interference effect in this system.     

Voltammetric studies of the interaction of rutin with DNA and its analytical applications on the MWNTs-COOH/Fe3O4 modified electrode

Multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH)/iron oxide (Fe₃O₄) modified glassy carbon electrode (MWNTs-COOH/Fe₃O₄/GCE) and DNA/MWNTs-COOH/Fe₃O₄/GCE were prepared. The electrochemical behaviors of rutin (RU) were investigated on MWNTs-COOH/Fe₃O₄/GCE by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in britton-robinson buffer solution (B-R). The interaction of RU with DNA was also explored. Dramatic decrease of peak current without obvious peak potential shift were observed in both cases of DNA in the solution and immobilized on the electrode surface. In addition, the electron transfer coefficient (α) and the rate constant (k_s) kept unchanged in the absence and presence of DNA. So interaction of DNA with RU formed a non-electroactive complex. The binding constant and binding ratio was obtained in the process. The interaction was also confirmed by UV-visible spectroscopy. The reduction peak current was linear with the concentration of RU in the range of 2.50 × 10⁻⁸ to 1.37 × 10⁻⁶ M, with a detection limit of 7.5 nM. The MWNTs-COOH/Fe₃O₄/GCE showed comparatively low detection limit, rapid response, simplicity for the determination of RU.     

Cu chemosensing behaviour of self-organized micro-array structures of a donor-acceptor bichromophoric compound anchored onto Ag nanoisland films

This work reports on the Cu²⁺ chemosensing behaviour of self-organized micro-array structures of a novel donor-acceptor bichromophoric compound anchored onto Ag nanoisland films. The system exhibits quenching of the fluorescence in the presence of Cu²⁺ ions, with detection range extending from 2 × 10⁻⁸ M up to 3 × 10⁻⁶ M and limit of detection (LOD) of 8 × 10⁻⁹ M. The quenching of fluorescence is accompanied by a quenching of SERS signal from the metal-organic structure, which is consistent with an electron transfer between the copper cation and the organic moiety. The self-organization property of the sensing complexes into micrometric arrays offers great potential for miniaturization and future development of Cu²⁺ detection systems based on real-time observation of fluorescence or SERS quenching by fluorescence microscopy or microRaman spectroscopy.     

Micromachined catalytic combustible hydrogen gas sensor

An integrated catalytic combustion H₂ sensor has been fabricated by using MEMS technology. Both the sensing elements and the reference elements could be integrated into the suspended micro heaters connected in a suitable circuit such as a Wheatstone configuration with low power consumption. Two sensitive elements and two reference sensors were integrated together onto a single chip. The size of chip was 5.76 mm² and the catalytic combustion sensor showed high response to H₂ at operating voltage of 1 V. The response and recovery times to 1000 ppm H₂ were 0.36 s and 1.29 s, respectively.     

Acetylene sensor based on Pt/ZnO thick films as prepared by flame spray pyrolysis

ZnO nanoparticles loaded with 0.2-2.0 at.% Pt have been successfully produced in a single step by flame spray pyrolysis (FSP) technique using zinc naphthenate and platinum(II) acetylacetonate, as precursors dissolved in xylene and their acetylene sensing characteristics have been investigated. The particle properties were analyzed by XRD, BET, TEM, SEM and EDS. Under the 5/5 (precursor/oxygen) flame condition, ZnO nanoparticles and nanorods were observed. The crystallite sizes of ZnO spherical and hexagonal particles were found to be ranging from 5 to 20 nm while ZnO nanorods were seen to be 5-20 nm in width and 20-40 nm in length. In addition, very fine Pt nanoparticles with diameter of ∼1 nm were uniformly deposited on the surface of ZnO particles. From gas-sensing characterization, acetylene sensing characteristics of ZnO nanoparticles is significantly improved as Pt content increased from 0 to 2  at.%. The 2 at.% Pt loaded ZnO sensing film showed an optimum C₂H₂ response of ∼836 at 1% acetylene concentration and 300 °C operating temperature. A low detection limit of 50 ppm was obtained at 300 °C operating temperature. In addition, Pt loaded ZnO sensing films exhibited good selectivity towards hydrogen, methane and carbon monoxide.     

SPR生物传感器的应用现状与发展趋势

表面等离子体共振(SPR)生物传感器是一种基于物理光学原理的新型生化分析系统.与传统的超速离心、荧光法相比,具有实时检测、无需标记、耗样量少等特点.介绍了SPR生物传感器的基本原理,着重介绍了SPR生物传感器在生命科学,药物残留,疾病诊断以及食品检测中的应用,并对其发展趋势进行了展望.     

Ethanol sensing using CuO/MWNT thin film

Copper (II) oxide (CuO)/multiwall carbon nanotube (MWNT) thin film based ethanol-sensors were fabricated by dispersing CVD-prepared MWNTs in varying concentration over DC magnetron sputtered-CuO films. The responses of these sensors as a function of MWNT concentrations and temperatures were measured, and compared. The sensing response was the maximum at an operating temperature near 400 °C for all the samples irrespective of the MWNTs dispersed over them. At optimum operating temperature (T_opt) of 407 ± 1 °C, the response is linear for 100-700 ppm range and tends to saturate at higher concentrations. In comparison with bare CuO sample, the response of CuO/MWNT sensing films increased up to 50% in the linear range. The response improvement for 2500 ppm of ethanol was up to 90% compared to bare CuO sample. In addition, the sensing response time also reduced to around 23% for lowest ethanol concentration at T_opt. However, a decrease in the sensor response was observed on films with very high concentrations of MWNTs.