Novel plastic biochips for colorimetric detection of biomolecules

We report a novel plastic biochip for the sensitive colorimetric detection of analytes of interest. This type of biochip is designed to perform bioassays in a sandwich format, i.e., employing the immobilized probe molecules to capture target and then utilizing gold nanoparticle (AuNP)-labeled reporters to screen the biorecognition events. To fabricate and implement such plastic biochips, not only have we demonstrated the probe immobilization, sensor unit formation, signal transduction and visualization on the plastic substrate, but we have also introduced new methods for imaging and analysis of them. As two proof-of-concept detection applications, plastic immunochips and DNA biochips have been fabricated and their responses to human IgG and DNA have been examined respectively. To further assess the detection sensitivity of the colorimetric-based biochip, we have compared it with an enzyme-catalyzed-based biochip and with a conventional fluorescent-based biochip. We believe the colorimetric-based plastic biochip presented herein is able to fully combine the advantage of colorimetric detection and plastic substrate, thus making it an ideal platform for point-of-care analysis and diagnostics.     

Fabrication and characterization of semicircular detection electrodes for contactless conductivity detector - CE microchips

This study uses simple and reliable microfabrication techniques to fabricate CE biochips, integrating a novel contactless conductivity detector in a miniaturized detection system in a microfluidic biochip. The off-channel electrodes are deposited around side channels by Au sputtering and patterned using a standard "lift-off" process. A vacuum fusion bonding process is employed to seal the lower substrate containing the microchannels and the electrodes to an upper glass cover plate. The variations in the capacitance between the semicircular detection electrodes in the side channels are measured as different samples and ions pass through the detection region of the CE separation channel. Samples of Rhodamine B, commercial sports drinks, mineral waters, and a red wine, respectively, are mixed in different buffer solutions, separated, and successfully detected using the developed device. The semicircular detection electrodes for the contactless conductivity detector have microscale dimensions and provide a valuable contribution to the realization of the lab-on-a-chip concept.     

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.     

A Review of Multifunctions of Dielectrophoresis in Biosensors and Biochips for Bacteria Detection

This paper reviews the functions of dielectrophoresis (DEP) that have been applied to biosensor and biochip platforms for bacteria detection, including concentration of bacterial cells from continuous flows, separation of target bacterial cells from non-target cells, as well as the enhancement of antibody capture efficiency on biosensor and biochip surfaces. DEP could provide effective concentration and separation simultaneously in well-designed microfluidic biosensor and biochip systems. The integration of DEP with a detection system allows the integration of sample preparation and enrichment steps with detection, which has the potential to eliminate the traditionally used time-consuming culture-based enrichment steps and other multiple off-chip sample preparation steps. DEP is also useful in biosensor and biochips platforms for enhancing antibody capture efficiency in both flow-through and non-flow-through microdevices. The enhanced antibody capture efficiency could allow the sensor capture more cells and to be detected by the sensor, particularly in dealing with low number of cells. The integration of multifunctions of DEP into biosensor and biochip platform has the potential to improve the detection of bacterial cells.     

Direct immobilization in poly(dimethylsiloxane) for DNA, protein and enzyme fluidic biochips

A new arraying method is presented based on the properties of poly(dimethylsiloxane) (PDMS) polymer to entrap beads bearing biologically active compounds. It is shown that such beads could be spotted and dried at the surface of a poly(vinyl chloride) master and subsequently transferred at the PDMS interface by direct moulding of the polymer on the mask. Moreover, the use of the PDMS-assisted-immobilization enables the development of either a low density array (100 spots) or a micro-channel biochip with a direct incorporation of the sensing element in a fluidic system for the quantitative detection of enzyme substrates, antigens and oligonucleotides, depending on the immobilized sensing element. All biochip formats were revealed by a chemiluminescent reaction detected with a charge coupled device camera.As a result, arrays of beads bearing active enzymes, antibodies and oligonucleotides were successfully obtained and enabled the achievement of biochips for the chemiluminescent detection of enzyme substrates, protein antigens and oligonucleotides sequence with detection limit of 1 μM, 1.5×10⁷ molecules and 10⁸ molecules, respectively.     

Biochip data normalization using multifunctional probes

Using a biochip with stable probe functionalization and a detection system capable of real time measurements, it is demonstrated that acquired probe-target interaction data are more reproducible in time--on a given probe spot using sequential target runs--than in space, using many probe spot replicates on the biochip in one single parallel target run. To increase the biochip data precision, a normalization method that quantifies and corrects the surface inhomogeneity without the use of complex data post-processing has been developed. This simple and effective method is based on adding a common reactive group to all probes and quantifying the biochip response to a calibration target, thus quantifying the spatial heterogeneity in the biosensor responsiveness. The usefulness of such methodology, which can be easily generalized, is demonstrated in the model case of DNA:DNA interactions, using a surface plasmon resonance imaging system as the dynamical reader. The biochips are based on streptavidin biochemically functionalized gold films onto which biotinylated ssDNA probe sequences, related to cystic fibrosis genotyping, are spotted. This normalization method provides high gain in data precision and allows, in this example, unambiguous genotyping of SNP, including discrimination of the heterozygote case from the two homozygote cases.     

Development of a respirometric biochip for embryo assessment

A prototype respirometric biochip dedicated to monitoring oxygen consumption of preimplantation embryos has been developed. The biochip comprises a linear array of eight flow-through microchambers profiled on silicon substrate, and functions together with a phosphorescent oxygen sensitive probe and fluorescence plate reader detection. A high level of sensitivity to changes in dissolved oxygen was achieved through miniaturisation and optimization of biochip geometry, and incorporation of appropriate sealing and humidification systems. The biochips have allowed characterisation of oxygen consumption, by 2 cell and blastocyst stage preimplantation mouse embryos, through monitoring as few as ten preimplantation embryos over a one-hour time period. They provide a non-invasive, simple and convenient means for assessing preimplantation embryo metabolism.     

Writing and reading methodology for biochips with sub-100-nm chemical patterns based on near-field scanning optical microscopy.

This paper demonstrates a writing and reading methodology, which allows both to create and to detect sub-100-nm carboxyl-terminated patterns on light-transmissive quartz substrates by the same instrumental system. Such a technique, capable of creating carboxyl-terminated nanopatterns, offers several benefits for the miniaturization of biochips, since the carboxyl-terminated nanopatterns allow the easy immobilization of biomolecules by amide bond formation. As a consequence, increasingly miniaturized biochips require suitable analytical methods for the detection of nanopatterns. In our approach, carboxyl-terminated nanopatterns of down to 80 nm width were created using a photolabile silane coupling agent and a UV laser coupled to a near-field scanning optical microscope (NSOM). The same NSOM system was then used in a next step to detect the fabricated carboxyl-terminated nanopatterns after modification with a fluorescent label. Furthermore, as a first step towards biochip applications, the successful immobilization of several biomolecules, such as streptavidin, IgG and DNA on carboxyl-terminated nanopatterns was demonstrated. We have shown that our approach has the potential to lead to a new bioanalytical method, which enables one to write and to read biochips on a sub-100-nm scale by the same system.     

Advanced Immobilization and Amplification for High Performance Protein Chips

With the success of high-throughput DNA microarrays, protein biochips have been intensively investigated and broadly used in bioscience research, clinic diagnosis, drug discovery, and other applications. However, there is great need to significantly improve the sensitivity of protein chips, especially in early diagnosis. A major challenge of improving sensitivity is that protein detection does not have an effective amplification method, such as PCR for DNA microarrays. Construction of unique biofilms for efficient immobilization of protein probes and innovation of new amplification schemes could play a critical role in performance improvement of protein biochips. With dramatic developments in microfabrication, nanotechnologies, and biotechnologies, enormous progress has been made, particularly in improving biosensing sensitivity. This article reviews new advances in protein biochip technologies with emphasis on novel approaches for efficient probe immobilization and nanomaterials-assisted signal amplification for high performance protein chips. Prominent progress in integration of protein microarrays with microfluidic platforms is briefly discussed. The major challenges and perspectives on the future of protein biochips are also addressed.     

彩色表面等离子体共振成像初探

自主设计并建立了彩色表面等离子共振成像(Color SPRI, CSPRI)实验系统, 并结合利用自己编制的软件开展了相关研究, 成功地观测到溶液和蛋白点阵的彩色图像. 这些结果显示, CSPRI有可能成为生物分子微点阵(或生物芯片)的一种新型的彩色显示手段.     

Regenerable immuno-biochip for screening ochratoxin A in green coffee extract using an automated microarray chip reader with chemiluminescence detection

Ochratoxin A (OTA) can contaminate foodstuffs in the ppb to ppm range and once formed, it is difficult to remove. Because of its toxicity and potential risks to human health, the need exists for rapid, efficient detection methods that comply with legal maximum residual limits. In this work we have synthesized an OTA conjugate functionalized with a water-soluble peptide for covalent immobilization on a glass biochip by means of contact spotting. The chip was used for OTA determination with an indirect competitive immunoassay format with flow-through reagent addition and chemiluminescence detection, carried out with the stand-alone automated Munich Chip Reader 3 (MCR 3) platform. A buffer model and real green coffee extracts were used for this purpose. At the present, covalent conjugate immobilization allowed for at least 20 assay-regeneration cycles of the biochip surface. The total analysis time for a single sample, including measurement and surface regeneration, was 12 min and the LOQ of OTA in green coffee extract was 0.3 μg L⁻¹ which corresponds to 7 μg kg⁻¹.     

Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction

In this report, Au-coated nanostructured biochip with functionalized thiolated primers on its surface is developed for label-free and real-time optical detection of polymerase chain reaction (PCR). A PCR chamber of 150 μm in thickness containing Au-coated nanostructured substrate in the bottom layer was bordered with SU-8 100 walls. After immobilization of 5′-thiolated primers on the surface, simultaneous DNA amplification and detection were performed without any labeled molecules via the relative reflected intensity (RRI) of Au-coated nanostructured substrate. When human genomic DNA at several concentrations of 0.2, 0.5 and 1 ng μL⁻¹ was included in the initial DNA samples, the increases in the RRI peak values were clearly observed with the increasing PCR cycle numbers. We found that the starting point of the optical signal, which was divergent from the background in our PCR biochip, was around 3-4 cycles, much lower than that of the fluorescent real-time PCR analysis (around 23-25 cycles). Our proposed PCR device using Au-coated nanostructured substrate holds noteworthy promise for rapid, label-free and real-time DNA detection for point-of-care testing (POCT) applications.     

Demonstration of a surface plasmon-coupled emission (SPCE)-based immunoassay in the absence of a spacer layer

The technique of surface plasmon-coupled emission (SPCE) involves the coupling of light which is emitted from a fluorophore into the surface plasmon of an adjacent thin metal film, giving rise to highly directional emission. We have combined the advantages of SPCE with the high light collection efficiency of supercritical angle fluorescence by carrying out an immunoassay on a paraboloid array biochip in the absence of the conventional SPCE spacer layer normally used to minimize metal quenching of the fluorescence. In this work, we have successfully demonstrated an SPCE-based assay by utilizing the protein assay layer as the spacer layer. A novel 3 × 3 injection molded polymer biochip with paraboloid elements was used. The paraboloid elements served to enhance the light collection efficiency while the top surface was coated with a gold layer to use excitation of surface plasmons and detection of SPCE emission. Theoretical modeling of the gold-protein layer structure showed that the surface plasmon resonance angles were located in the detection range of the paraboloid biochip. The polarization dependence of SPCE emission was also demonstrated. Finally, a human IgG sandwich immunoassay was carried out which exhibited a limit of detection of ~10 ng/ml using 3σ. The results demonstrate the potential of the SPCE-based paraboloid array biochip as a novel platform for high-throughput analysis of biomolecular interactions.     

A Direct Electrochemical Detection Method of Melanoma Based on Melanoma Biomarker

A new method of detecting and diagnosing melanoma based on melanoma biomarker was developed and its feasibility demonstrated. The method is based on an electrochemical biosensor platform comprised of a special biochip and device, performing a multi‐channel amperometric detection of the enzymatic activity of tyrosinase, an enzyme biomarker of melanoma. The newly developed biosensor platform is able to electrochemically detect tyrosinase activity in fresh biopsy samples. This bioelectrochemical detection method is rapid, yielding results within minutes from biopsy removal. Using “as is” biopsy samples, without pretreatment, simplifies the process, saves time and reduces cost and labor dramatically. Using modern portable microelectronics provides an accurate biomarker expression measurement at the “point of care” increasing the accessibility of new bio‐chip technologies to the public.     

Rapid detection of single cell bacteria as a novel approach in food microbiology

Solid-phase cytometry (SPC) is a novel technique that allows rapid detection of bacteria at the single cell level, without the need for a growth phase. After filtration of the sample, the retained microorganisms are fluorescently labeled on the membrane filter and automatically counted by a laser scanning device. Each fluorescent spot can be visually inspected with an epifluorescence microscope connected to the ChemScan by a computer-driven moving stage. Depending on the fluorogenic labels used, information on the identity and the physiological status of the microorganisms can be obtained within a few hours. Although SPC was originally recommended for the determination of the total viable microbial count in water and other liquid samples, it may also be a promising technique for the detection and enumeration of bacteria in food samples, provided they can be isolated from the unfilterable matrix. The short detection time inherent in this approach is a considerable advantage over conventional plate counting, especially for slow-growing microorganisms. The basic principles of SPC are discussed as well as its potential for the detection of Mycobacterium paratuberculosis, a model example of a slow-growing bacterium in milk.     

Surface plasmon resonance application in prostate cancer biomarker research

Prostate cancer (PCa) diagnostics can be effectively addressed using sensor-based approaches. Proper selection of biomarkers to be included in biosensors for accurate detection becomes the need of the hour. Such biosensor and biochip technologies enable fast and efficient determination of proteins and provide a remarkable insight into the changes in the protein structure, such as aberrant glycosylation, which can increase the performance, sensitivity and specificity of clinic assays. However, for a thorough comprehension of such complex protein modifications, it is crucial to understand their biospecific interactions. Surface plasmon resonance (SPR), one of the most rapidly developing techniques for measuring real-time quantitative binding affinities and kinetics of the interactions of antigens and antibodies, was chosen as an appropriate tool for this purpose. Herein, experiments on the interactions of antibodies specific against different epitopes of free and complexed prostate-specific antigen (PSA), a prominent PCa biomarker, are presented with two main aims: (i) to continue as lectin glycoprofiling studies and; (ii) to be used in microfluidic immunoassay-based platforms for point-of-care devices. Various PSA-specific antibodies were covalently immobilized on the biochip surface via amine coupling, and free or complexed PSA was injected into the dual-flow channels of the SPR device. Kinetic parameters and affinity constants of these interactions, as well as cross-reactivities of the used antibodies were determined. The sandwich assay for PSA determination was developed employing both primary and secondary anti-PSA antibodies. Sensitivity of the assay was 3.63 nM^?1, the detection limit was 0.27 nM and the SPR biosensor response towards free PSA was linear up to 25 nM. All these findings are essential for proper design of a selective, sensitive, and highly reliable biosensor for PCa diagnosis as a lab-on-chip device.     

Microbial biosensors

A microbial biosensor is an analytical device that couples microorganisms with a transducer to enable rapid, accurate and sensitive detection of target analytes in fields as diverse as medicine, environmental monitoring, defense, food processing and safety. The earlier microbial biosensors used the respiratory and metabolic functions of the microorganisms to detect a substance that is either a substrate or an inhibitor of these processes. Recently, genetically engineered microorganisms based on fusing of the lux, gfp or lacZ gene reporters to an inducible gene promoter have been widely applied to assay toxicity and bioavailability. This paper reviews the recent trends in the development and application of microbial biosensors. Current advances and prospective future direction in developing microbial biosensor have also been discussed.     

Construction of hybrid bilayer membrane (HBM) Biochips and characterization of the cooperative binding between cytochrome-c and HBM

We constructed multi-channel hybrid bilayer membrane (HBM) biochips and characterized them by surface plasmon resonance imaging. Each channel in the biochip was prepared using vesicles with different proportions of negative, neutral, and positive lipids. The HBM surfaces were tested by interaction with two globular proteins that recognize surfaces covered with opposite charges. Spots modified with the same HBM show responses within a relative standard deviation of 10% or smaller. These devices were also used to study in detail the interaction between cytochrome-c (cyt-c) and HBMs. Cooperative binding between cyt-c and negative HBMs was demonstrated. Using an adaptation of the Hill model, we calculated a Hill coefficient of 5 and a 10-fold increase in the binding constant with the increase in cyt-c concentration. We propose that this treatment can be used to evaluate the cooperative binding of surface proteins to membranes.     

Latex bead immobilisation in PDMS matrix for the detection of p53 gene point mutation and anti-HIV-1 capsid protein antibodies

Two diagnostic chemiluminescent biochips were developed for either the detection of p53 gene point mutation or the serological detection of anti-HIV-1 p24 capsid protein. Both biochips were composed of 24 microarrays of latex beads spots (4×4) (150 m in diameter, 800 m spacing) entrapped in a poly(dimethylsiloxane) elastomer (PDMS). The latex beads, bearing oligonucleotide sequences or capsid protein, were spotted with a conventional piezoelectric spotter and subsequently transferred at the PDMS interface. The electron microscopy observation of the biochips showed how homogeneous and well distributed the spots could be. Point mutation detection on the codon 273 of the p53 gene was performed on the basis of the melting temperature difference between the perfect match sequence and the one base pair mismatch sequence. The hybridisation of a 20-mer oligonucleotide form the codon 273 including a one base pair mutation in its sequence on a biochip arrayed with non-muted and the muted complementary sequences, enabled a clear discrimination at 56°C between muted and wild sequences. Moreover, the quantitative measurement of the amount of muted sequence in a sample was possible in the range 0.4–4 pmol. Serological measurement of anti-HIV-1 p24 capsid protein on the biochip, prepared with 1-m-diameter latex beads, enabled the detection of monoclonal antibodies in the range 1.55–775 ng mL^–1. Such a range could be lowered to 0.775 ng mL^–1 when using 50-nm-diameter beads, which generated a higher specific surface. The validation of the biochip for the detection of anti-HIV-1 capsid protein antibodies was performed in human sera from seropositive and seronegative patients. The positivity of the sera was easily discriminated at serum dilutions below 1:1,000.     

Quantitation of surface-bound proteins on biochips using MALDI-TOF MS

We report on a novel method for the quantitation of proteins specifically bound on a ligand-presenting biochip by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The bound protein was digested by trypsin, and the resulting peptide fragments were analyzed by MALDI-TOF MS in the presence of an isotope-labeled internal standard (IS). The IS has the same sequence as a reference peptide (RP) of the target protein digest, but a different molecular weight. The absolute amount of the specifically bound protein on a biochip is then quantitated by comparison of mass intensities between the RP and the IS. Because they have the same molecular milieu, the mass intensities of these two analytes represent the real amounts of analytes on the chip. As a model system, we tested glutathione s-transferase (GST) and a GST-fusion protein, which were captured on glutathione-presenting biochips. We observed that the glutathione densities on biochips showed a good correlation with the absolute quantity of the proteins. We believe that our method will provide an alternative to currently existing tools for the absolute quantitation of surface-bound proteins.