Ultrasensitive electrical detection of nucleic acids by hematin catalysed silver nanoparticle formation in sub-microgapped biosensors

An ultrasensitive electrical detection method of nucleic acids has been demonstrated on sub-microgapped biosensor. In this method, peptide nucleic acid (PNA) probes were firstly immobilized in the gap areas of a pair of interdigited microelectrodes and then were hybridized with their complementary target DNA. After hybridization, hematin molecules were introduced into the DNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hematin molecules act as a catalyst to accelerate reducing ammoniacal silver ion to form silver nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the silver nanoparticles directly correlated with the number of the hybridized DNA molecules. Nearly 1fM sensitivity was achieved under optimal conditions. This approach is also applicable to the detection of RNA.     

Current based antibodies detection from human serum enhanced by secondary antibodies labelled with gold nanoparticles immobilized in a nanogap

We present the electrical detection of immunoglobulin G (IgGs) from human serum using a nanogap-based biosensor. The detection method is based on the capture of IgGs by a probe immobilized between gold nanoelectrodes of 30-90nm spacing. The captured IgGs are further reacted with secondary antibodies labelled with gold nanoparticles (GNPs). Insertion of GNPs into the nanogap resulted in increasing the conductance through the nanogap. The use of a chip with 90 nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specific and differential capture of human antibodies by arrayed probes. We obtained a 500-fold higher quality factor with protein A compared to goat anti-murine antibodies. This method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples.     

Detection of proteins and bacteria using an array of feedback capacitance sensors

An integrated array of micron-dimension capacitors, originally developed for biometric applications (fingerprint identification), was engineered for detection of biological agents such as proteins and bacteria. This device consists of an array of 93,184 (256 x 364) individual capacitor-based sensing elements located underneath a thin (0.8 microm) layer of glass. This glass layer can be functionalized with organosilane-based monolayers to provide groups amenable for the immobilization of bioreceptors such as antibodies, enzymes, peptides, aptamers, and nucleotides. Upon functionalization with antibodies and in conjunction with signal amplification schemes that result in perturbation of the dielectric constant around the captured antigens, this system can be used as a detector of biological agents. Two signal amplification schemes were tested in this work: one consisted of 4 microm diameter latex immunobeads and a second one was based on colloidal gold catalyzed reduction of silver. These signal amplification approaches were demonstrated and show that this system is capable of specific detection of bacteria (Escherichia coli) and proteins (ovalbumin). The present work shows proof-of-principle demonstration that a simple fingerprint detector based on feedback capacitance measurements can be implemented as a biosensor. The approach presented could be easily expanded to simultaneously test for a large number of analytes and multiple samples given that this device has a large number of detectors. The device and required instrumentation is highly portable and does not require expensive and bulky instrumentation because it relies purely on electronic detection.     

Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3alpha-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10-80 microM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5-30 microM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.     

Using an electro-microchip, a nanogold probe, and silver enhancement in an immunoassay

This paper presents a novel immunoassay that uses an electro-microchip to detect the immuno-reaction signal, gold nanoparticles (ANPs) as a label of antigen or antibody and as a catalyst for silver precipitation, and the silver enhancement reaction to magnify the detection signal. This study is based on the direct immunoassay (two-layer format) and the sandwich immunoassay (three-layer format). The ANPs were introduced into the electro-microchip by the specific binding of the antibodies-ANPs conjugates and then were coupled with silver enhancement to produce black spots of silver metal. The silver precipitation constructs a "bridge" between two electrodes of the electro-microchip allowing electrons to pass. The variation of impedance can be easily measured with a commercial LCR meter. Various gap sizes (20, 50, 100, and 200 microm) of the electrodes of electro-microchips were designed for the sensitivity study. The experimental data show that a chip with a 20microm gap has the highest sensitivity. There was a significant difference in impedance between the experiment sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than the conventional enzyme-linked immunosorbent assay (ELISA). In addition, it shows a high detection sensitivity (10 microg/mL of 1st antibody (IgG) immobilized on slides and 1 ng/mL of antigen (protein A)). There is a clear distinction between the signal intensity and the logarithm of the sample concentration. The proposed new immunoassay method has potential applications in proteomics research and clinical diagnosis.     

Label-free immunosensor based on gold nanoparticle silver enhancement

A label-free immunosensor for the sensitive detection of human immunoglobulin G (IgG) was prepared based on gold nanoparticle-silver enhancement detection with a simple charge-coupled device (CCD) detector. The gold nanoparticles, which were used as nuclei for the deposit of metallic silver and also for the adsorption of antibodies, were immobilized into wells of a 9-well chip. With the addition of silver enhancement buffer, metallic silver will deposit onto gold nanoparticles, causing darkness that can be optically measured by the CCD camera and quantified using ImageJ software. When antibody was immobilized onto the gold nanoparticles and antigen was captured, the formed immunocomplex resulted in a decrease of the darkness and the intensity of the darkness was in line with IgG concentrations from 0.05 to 10 ng/ml. The CCD detector is simple and portable, and the reported method has many desirable merits such as sensitivity and accuracy, making it a promising technique for protein detection.     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

Nanoparticle-based electrochemical detection of hepatitis B virus using stripping chronopotentiometry

A selective and sensitive gold nanoparticle-based electrochemical method for detection of hepatitis B virus DNA sequences was used. This method relies on the hybridization of amplified hepatitis B virus DNA strands with probes that are extended on paramagnetic beads. After separation of noncomplementary sequences, hybridized magnetic beads were treated with streptavidin-modified gold followed by silver enhancement. High selectivity and high sensitivity were obtained using electrochemical stripping detection of silver ions that were deposited on gold nanoparticles. With a signal/noise ratio of approximately 4.6, the detection limit was estimated to be 0.7ng/ml.     

A signal amplification assay for HSV type 1 viral DNA detection using nanoparticles and direct acoustic profiling

Background  

Nucleic acid based recognition of viral sequences can be used together with label-free biosensors to provide rapid, accurate confirmation of viral infection. To enhance detection sensitivity, gold nanoparticles can be employed with mass-sensitive acoustic biosensors (such as a quartz crystal microbalance) by either hybridising nanoparticle-oligonucleotide conjugates to complimentary surface-immobilised ssDNA probes on the sensor, or by using biotin-tagged target oligonucleotides bound to avidin-modified nanoparticles on the sensor. We have evaluated and refined these signal amplification assays for the detection from specific DNA sequences of Herpes Simplex Virus (HSV) type 1 and defined detection limits with a 16.5 MHz fundamental frequency thickness shear mode acoustic biosensor.     

Enhanced Sensitivity of Surface Plasmon Resonance Phase-Interrogation Biosensor by Using Silver Nanoparticles

It is demonstrated that the sensitivity of surface plasmon resonance phase-interrogation biosensor can be enhanced by using silver nanoparticles. Silver nanoparticles were fabricated on silver films by using thermal evaporation. Sizes of silver nanoparticles on silver thin film can be tuned by controlling the deposition parameters of thermal evaporation. By using surface plasmon resonance heterodyne interferometey to measure the phase difference between the p and s polarization of incident light, we have demonstrated that sensitivity of glucose detection down to the order of 10⁻⁸ refractive index units can be obtained.     

Nanowire labeled direct-charge transfer biosensor for detecting Bacillus species

A direct-charge transfer (DCT) biosensor was developed for the detection of the foodborne pathogen, Bacillus cereus. The biosensor was fabricated using antibodies as the sensing element and polyaniline nanowire as the molecular electrical transducer. The sensor design consisted of four membrane pads, namely, sample application, conjugate, capture and absorption pads. Two sets of polyclonal antibodies, secondary antibodies conjugated with polyaniline nanowires and capture antibodies were applied to the conjugate and the capture pads of the biosensor, respectively. The detection technique was based on capillary flow action which allowed the liquid sample to move from one membrane to another. The working principle involved antigen-antibody interaction and direct electron charge flow to generate a resistance signal that was being recorded. Detection from sample application to final results was completed in 6 min in a reagentless process. Experiments were conducted to find the best performance of the biosensors by varying polyaniline types and concentrations. Polyaniline protonated with hydrochloric acid, emeraldine salt and polyaniline protonated with perchloric acid were the three kinds of polyaniline used in this study. The biosensor sensitivity in pure cultures of B. cereus was found to be 10(1) to 10(2)CFU/ml. Results indicated that using emeraldine salt at a concentration of 0.25 g/ml gave the best biosensor performance in terms of sensitivity. The biosensor was also found to be specific in detecting the presence of B. cereus in a mixed culture of different Bacillus species and other foodborne pathogens. The speed, sensitivity and ease-of-use of this biosensor make it a promising device for rapid field-based diagnosis towards the protection of our food supply chain. The phenotypic and genotypic similarities between B. cereus and Bacillus anthracis will also allow this biosensor to serve as an excellent model for the detection of B. anthracis.     

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.     

SOI-nanowire biosensor for detection of D-NFATc1 protein

The nanowire (NW) detection is one of the fast-acting and high-sensitive methods, which can recognize potentially relevant protein molecules. A NW-biosensor based on the silicon-on-insulator (SOI)-structures has been used for biospecific label-free real time detection of the NFATc1 (D-NFATc1) oncomarker. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFATc1 used as molecular probes. It was shown that using this biosensor it is possible to reach sensitivity of 10^?15 M. This sensitivity was comparable to that of the NW-biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate that approaches used in this study are promising for development of sensor elements for high-sensitive diagnostics of diseases.     

Label-free antibody-antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles

Gold nanoparticles grown in situ from printed seed particles on a glass substrate have been fabricated into a biosensor array. The light-scattering properties of the resulting surfaces show sensitivity to changes in the local refractive index. Each array spot is functionalized with fibrinogen or bovine serum albumin and scattered radiation is used to monitor the refractive index change on label-free binding of the antibodies to their antigens from whole blood antiserum. Data were collected real-time and the association rate constants for the specific antibody-antigen binding were derived from a kinetic analysis. The minimum antibody concentration detection sensitivity is of 100 nM.     

On-chip manipulation and detection of magnetic particles for functional biosensors

We demonstrate the real-time on-chip detection and manipulation of single 1 microm superparamagnetic particles in solution, with the aim to develop a biosensor that can give information on biological function. Our chip-based sensor consists of micro-fabricated current wires and giant magneto resistance (GMR) sensors. The current wires serve to apply force on the particles as well as to magnetize the particles for on-chip detection. The sensitivity profile of the sensor was reconstructed by simultaneously measuring the sensor signal and the position of an individual particle crossing the sensor. A single-dipole model reproduces the measured sensitivity curve for a 1 microm bead. For a 2.8 microm bead the model shows deviations, which we attribute to the fact that the particle size becomes comparable to the sensor width. In the range between 1 and 10 particles, we observed a linear relationship between the number of beads and the sensor signal. The real-time detection and manipulation of individual particles opens the possibility to perform on-chip high-parallel single-particle assays.     

Simple detection of nucleic acids with a single-walled carbon-nanotube-based electrochemical biosensor

We report for the first time a simple approach to fabricate an electrochemical DNA (E-DNA) biosensor by introducing the single-walled carbon nanotubes (SWNTs). The SWNTs combine with the electrochemical label (methyl blue, MB)-modified single-stranded DNA (ssDNA) probes to generate a nanomaterial-biomolecule composite, which functions as a signal amplification platform to facilitate the electron-transfer between the electrochemical label and the electrode. This SWNT-based E-DNA biosensor produces a high square wave voltammetry (SWV) signal in the absence of target DNA. In the presence of target DNA, the MB-labeled ssDNA probes are removed from the SWNT-modified electrode due to the formation of a double-stranded DNA (dsDNA), generating a relatively low SWV signal. This signal-off SWNT-based E-DNA biosensor exhibits improved sensitivity and large linear dynamic range with low detection limit; it can even distinguish 1-base mismatched target DNA. Further experiments demonstrate that the SWNT-based E-DNA biosensor is superior to the multi-walled carbon nanotube (MWNT)-based one for DNA detection. Moreover, the introduction of aptamer into the SWNT-based biosensor might be further extended to detect small biomolecules such as adenosine.     

Performance enhancement of polyaniline-based polymeric wire biosensor

We demonstrate here the performance enhancement of polyaniline-based biosensor using screen-printing technology and pulse mode measurement technique. Screen-printed silver electrodes were made on a nitrocellulose membrane and the distance between the two electrodes was approximately 550 microm. Resistance of the electrodes had an average of 1.4 Omega with a standard deviation of +/-0.4 Omega. The surface of nitrocellulose membrane was modified by glutaraldehyde to immobilize streptavidin. Biotinylated anti-mouse IgG was conjugated with polyaniline-coated magnetic nanoparticles. Formation of polyaniline-coated magnetic nanoparticles was confirmed by a transmission electron microscope image. The polyaniline was used as an electric signal transducer for the monitoring of the biospecific binding event. An electrical response induced by the streptavidin-biotin interaction was measured by pulse mode measurement. This measurement method reduced the resistance caused by interfacial capacitance. Dose-dependent resistance changes were also successfully analyzed by the pulse mode polymeric wire biosensor. Results showed that the pulse mode measurement technique enhanced the performance of the polyaniline-based polymeric wire biosensor by reducing the interfacial effects. This approach could be helpful in samples with high interfering background materials, such as food and clinical specimens.     

纳米金标记-逐步银染法快速检测金黄色葡萄球菌

将纳米金探针应用于目的核酸的检测,具有与PCR相当的灵敏度和特异性.本研究建立了一种可以在微孔板上快速检测金黄色葡萄球菌的纳米金标记-逐步银染法.该方法利用已包被链霉亲和素的微孔板,将PCR扩增的金黄色葡萄球菌nuc基因与生物素探针、纳米金探针形成的三明治杂交结构锚定其上,然后在低温下逐步银染显色,通过酶标仪检测放大的银染信号.这种纳米金标记-逐步银染法可以在显著降低非特异性背景信号的同时放大银染信号,检测金黄色葡萄球菌nuc基因的灵敏度为1 pmol/L,比常温一步银染法的灵敏度提高约102倍. 51例临床标本的检测结果与PCR法一致,与培养生化鉴定法的检测结果之间无显著性差异(P >0.05). 综上所述,本研究成功构建了金黄色葡萄球菌的纳米金标记-逐步银染法,在病原微生物的快速检测领域表现出广阔的发展潜力.     

Localized surface plasmon coupled fluorescence fiber-optic biosensor for alpha-fetoprotein detection in human serum

In this study, we demonstrated that the fiber-optic biosensor based on localized surface plasmon coupled fluorescence (LSPCF) is capable of detecting alpha-fetoprotein (AFP) in human serum. The sensitivity of LSPCF fiber-optic biosensor is not only enhanced but also the specific selectivity is improved since the fluorophores are excited by the localized surface plasmon with high efficiency. Experimentally, this fiber-optic biosensor is able to detect AFP concentration in phosphate buffered saline (PBS) solution from 0.1ng/mL to 100ng/mL whereas the linear relationship between the AFP concentrations and the fluorescence signals is shown. Furthermore, a linear response between the fluorescence signals and the concentrations of AFP in human serum from 2.33ng/mL to 143.74ng/mL is also obtained. As a result, the detection limit of the LSPCF fiber-optic biosensor on AFP detection is comparable with the conventional enzyme-linked immunosorbent assay (ELISA). Additionally, the LSPCF fiber-optic biosensor benefits on inexpensive, disposable and simpler optical geometry that can become a high efficient immunoassay comparable with the conventional ELISA and radioimmunoassay (RIA) clinically.     

Capillary-driven multiparametric microfluidic chips for one-step immunoassays

A new zinc oxide nanoparticles/chitosan/carboxylated multiwall carbonnanotube/polyaniline (ZnO-NPs/CHIT/c-MWCNT/PANI) composite film has been synthesized on platinum (Pt) electrode using electrochemical techniques. Three enzymes, creatinine amidohydrolase (CA), creatine amidinohydrolase (CI) and sarcosine oxidase (SO) were immobilized on ZnO-NPs/CHIT/c-MWCNT/PANI/Pt electrode to construct the creatinine biosensor. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The enzyme electrode detects creatinine level as low as 0.5 μM at a signal to noise ratio of 3 within 10s at pH 7.5 and 30°C. The fabricated creatinine biosensor showed linear working range of 10-650 μM creatinine with a sensitivity of 0.030 μA μM(-1)cm(-2). The biosensor shows only 15% loss of its initial response over a period of 120 days when stored at 4°C. The fabricated biosensor was successfully employed for determination of creatinine in human blood serum.