Systems biology advocates the understanding of biology at the systems-level, which requires massive information of correlations among individual components in complex biological systems. Such comprehensive investigation entails the use of high-throughput analytical tools. Microfluidic technology holds high promise to facilitate the progress of biology by enabling miniaturization and upgrading of current biological research tools due to its advantages such as low sample consumption, reduced analysis time, high-throughput and compatible sizes with most biological samples. In this article, we documented the recent applications of microfluidic chips in biological researches at the molecular level, cellular level and organism level, serving the purpose for systems-level understanding of biology. 相似文献
Here we report the development of a programmable and fully automatic gold array-embedded gradient microfluidic chip that integrates a gradient microfluidic device with gold-patterned microarray wells. This device provides a convenient and reproducible surface-enhanced Raman scattering (SERS)-based immunoassay platform for cancer biomarkers. We used hollow gold nanospheres (HGNs) as SERS agents because of their highly sensitive and reproducible characteristics. The utility of this platform was demonstrated by the quantitative immunoassay of alpha-fetoprotein (AFP) model protein marker. Our proposed SERS-based immunoassay platform has many advantages over other previously reported SERS immunoassay methods. The tedious manual dilution process of repetitive pipetting and inaccurate dilution is eliminated with this process because various concentrations of biomarker are automatically generated by microfluidic gradient generators with N cascade-mixing stages. The total assay time from serial dilution to SERS detection takes less than 60 min because all of the experimental conditions for the formation and detection of immunocomplexes can be automatically controlled inside the exquisitely designed microfluidic channel. Thus, this novel SERS-based microfluidic assay technique is expected to be a powerful clinical tool for fast and sensitive cancer marker detection. 相似文献
Low cost and scalable manufacture of lab-on-chip devices for applications such as point-of-care testing is an urgent need. Weaving is presented as a unified, scalable and low-cost platform for the manufacture of fabric chips that can be used to perform such testing. Silk yarns with different properties are first selected, treated with the appropriate reagent solutions, dried and handloom-woven in one step into an integrated fabric chip. This platform has the unique advantage of scaling up production using existing and low cost physical infrastructure. We have demonstrated the ability to create pre-defined flow paths in fabric by using wetting and non-wetting silk yarns and a Jacquard attachment in the loom. Further, we show that yarn parameters such as the yarn twist frequency and weaving coverage area may be conveniently used to tune both the wicking rate and the absorptive capacity of the fabric. Yarns optimized for their final function were used to create an integrated fabric chip containing reagent-coated yarns. Strips of this fabric were then used to perform a proof-of-concept immunoassay with sample flow taking place by capillary action and detection being performed by a visual readout. 相似文献
A universal sandwich-like immunoassay strategy based on quantum-dots immunoprobe (QD-labeled anti-mouse IgG antibody) was developed for rapid and ultrasensitive detection of small molecules. A portable and reusable optofluidic nano-biosensing platform was applied to investigate the sandwich-like immunoassay mechanism and format of small molecules, as well as the binding kinetics between QD immunoprobe and anti-small molecule antibody. A two-step immunoassay method that involves pre-incubation mixture of different concentration of small molecule and anti-small molecule antibody, and subsequent introduction of QD immunoprobe into the optofluidic cell was conducted for small molecule determination. Compared with the one-step immunoassay method, the two-step immunoassay method can obtain higher fluorescence signal and higher sensitivity index, thus improving the nano-biosensing performance. Based on the proposed strategy, two mode targets, namely, microcystin-LR (MC-LR) and Bisphenol A (BPA) were tested with high sensitivity, rapidity, and ease of use. A higher concentration of small molecules in the sample led to less anti-small molecule antibody bound with antigen-carrier protein conjugate immobilized onto the sensor surface, and less QD immunoprobes bound with anti-small molecule antibody. This phenomenon lowered the fluorescence signal detected by nano-biosensing platform. Under optimal operating conditions, MC-LR and BPA exhibited a limit of detection of 0.003 and 0.04 μg/L, respectively. The LODs were better than those of the indirect competitive immunoassay method for small molecules via Cy5.5-labeled anti-small molecule antibody. The proposed QD-based sandwich-like immunoassay strategy was evaluated in spiked water samples, and showed good recovery, precision and accuracy without complicated sample pretreatments. All these results demonstrate that the new detection strategy could be readily applied to the other trace small molecules in real water samples. 相似文献
An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001–0.1 ng mL−1 with a low detection limit down to 0.52 pg mL−1. The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system. 相似文献
Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies. 相似文献
Most cancers developed an elevation of the level of at least two markers associated with their incidence. Simultaneous detection
of multi-tumor markers associated with a particular type of cancer plays an important role in cancer diagnostic. Here, a multianalyte
immunoassay chip for simple and sensitive detection of tumor markers with chemiluminescent and colorimetric methods was proposed,
in which carcinoembryonic antigen (CEA) and carbohydrate antigen (CA19-9) that associated with colorectal cancer were detected
as model. The immunoassay chip was fabricated by co-immobilization of CEA/CA19-9 antibody on a glass slide with γ-glycidoxypropyltrimethoxysilane
as linkage. Through sandwiched immunoreactions, CEA, CA19-9, and their corresponding enzyme tracers, alkaline phosphatase-labeled
anti-CEA and horseradish peroxidase-labeled anti-CA19-9, were introduced on the chip. Then, they were sequentially detected
by chemiluminescent method in the range of 0.5–80 μg/L and 0.5–80 kU/L with the detection limits of 0.41 μg/L and 0.36 kU/L
at 3σ for CEA and CA19-9, respectively. They could also be detected by colorimetric method in the range of 1–200 μg/L and
5–200 kU/L with the detection limits of 0.25 μg/L and 1.25 kU/L at 3σ for CEA and CA19-9, respectively. All these results
demonstrated that the present work provided a promising analytical method for tumor markers’ analysis with the advantages
of simple analytical procedure, small sample volume and lower cost, which made the proposed method potential for high-throughput
detection. 相似文献
Influenza virus RNA was amplified by a continuous-flow polydimethylsiloxane microfluidic RT-PCR chip within 15-20 min. The amplified influenza virus RNA was observed with the naked eye, as the red color at the test line, using a lateral flow immunoassay within 1 min. 相似文献
A simple, reusable and ultrasensitive electrochemical clinic immunoassay is proposed via developing a versatile CTDONG (conductive three-dimensional ordered nano-gold) film-modified gold electrode, in which ferrocene derivative and human immunoglobulin G (hIgG) are used as signaling probe and model molecule, respectively. A signal-on signaling mechanism is achieved by utilizing a sandwich format of the primary antibody/hIgG/the secondary antibody labeled with Ferrocene (PAb/Ag/SAb). Owing to the combination of the advantages of CTDONG film with the versatility of ferrocene derivatives, a substantially enhanced signal accompanied by a low background peak current is achieved. By this sensing scheme, target molecule can be readily quantified in a comparatively wide dynamic range (8.1 × 10−13-6.2 × 10−10 M) with a relatively low detection limit (2.7 × 10−13 M). In addition to a greatly improved signal gain, this immunosensor gives a favourable reusability and good reproducibility. Moreover, the CTDONG film-based sensing interface shows excellent anti-interference ability to the coexistent proteins. Meanwhile, the recovery test and determination of target molecule in real samples have confirmed the feasibility of designed sensing system for clinic immunoassay of protein molecules, demonstrating the potential application of described CTDONG film in the development of biomolecule assay platforms. 相似文献
We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously. 相似文献
A strategy for design of a derivative of chlorsulfuron, which mimics half of the herbicide molecule, was proposed. The 1-[(2-chloro)phenylsulfonyl]monoamidosuccinic acid was synthesized as a derivative of chlorsulfuron for conjugation to carrier proteins. Rabbits were immunized and the resulting polyclonal antibodies were assessed by the fluorescence polarization technique. The antibodies were highly specific to chlorsulfuron. Cross-reactivity to the structurally similar sulfonylurea and urea herbicides chlorbromuron, amidosulfuron, chlortoluron, isoproturon, diuron and linuron was less than 0.1%. A rapid fluorescence polarization immunoassay (FPIA) for chlorsulfuron detection in water samples was developed and optimized. The detection limit of chlorsulfuron in 50 μl of sample was 10 ng ml−1. Total time for the measurement of 10 samples is 7 min. The proposed FPIA is suitable for rapid testing for pesticide contamination where the highest sensitivity is not critical or in combination with pre-concentration techniques. 相似文献
A simple, fast, efficient, and reusable microwave-assisted tryptic digestion system which was constructed by immobilization of trypsin onto porous core-shell Fe3O4@fTiO2 microspheres has been developed. The nanostructure with magnetic core and titania shell has multiple pore sizes (2.4 and 15.0 nm), high pore volume (0.25 cm3 g−1), and large surface area (50.45 m2 g−1). For the proteins, the system can realize fast and efficient microwave-assisted tryptic digestion. Various standard proteins (e.g., cytochrome c (cyt-c), myoglobin (MYO), β-lactoglobulin (β-LG), and bovine serum albumin (BSA)) used can be digested in 45 s under microwave radiation, and they can be confidently identified by mass spectrometry (MS) analysis; even the concentration of substrate is as low as 5 ng μL−1. Furthermore, the system for the 45 s microwave-assisted tryptic digestion is still effective after the trypsin-immobilized microspheres have been reused for 5 times. Importantly, 1715 unique proteins from 10 μg mouse brain proteins can be identified with high confidence after treatment of 45 s microwave-assisted tryptic digestion. 相似文献
A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR). Several physicochemical parameters such as the chemiluminescent assay mediums, the dilution ratio of MC-LR-OVA conjugate, monoclonal antibody concentration, and peroxidase labeled antibody concentration were studied and optimized. Under optimum conditions, calibration curve obtained for MC-LR had detection limits of 0.032 ± 0.003 μg L−1, the 50% inhibition concentration (IC50) was 0.20 ± 0.02 μg L−1 and the quantitative detection range was 0.062-0.65 μg L−1. The proposed methods was successfully applied to the monitoring of MC-LR in spiked water samples without significant effect of the matrix, and the recovery of MC-LR added to water samples at different concentrations ranged from 80% to 115% with the coefficients of variation (CVs) less than 9%. The LOD attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of CLEIA as a screening tool for the analysis of MC-LR in environmental samples. 相似文献
The use of PSU‐Py prepared by click chemistry as a platform in membrane‐bottom microwell plates for oxidase and hydrolase/oxidase‐based enzyme assays is studied. For the GOx assay, the postulated fluorescence mechanism is based on the consumption of glucose by dissolved oxygen and GOx in the microwell plates covered with the PSU‐Py membrane. For the AG‐GOx assay, maltose is used as AG substrate and hydrolyzed to glucose which is then oxidized by the GOx activity. It is shown that the PSU‐Py membrane acts as a fluorescence indicator of the enzymatic reactions, and both GOx and AG/GOx enzyme assays are successfully applied for glucose, maltose and acorbose analysis in the range 0.125–2.0 × 10?3 M glucose, 0.05–0.5 × 10?3 M maltose, and 0.0125–0.1 mg · mL?1 acorbose, respectively.
Current contaminant and residue monitoring throughout the food chain is based on sampling, transport, administration, and analysis in specialized control laboratories. This is a highly inefficient and costly process since typically more than 99 % of the samples are found to be compliant. On-site simplified prescreening may provide a scenario in which only samples that are suspect are transported and further processed. Such a prescreening can be performed using a small attachment on a cellphone. To this end, a cellphone-based imaging platform for a microsphere fluorescence immunoassay that detects the presence of anti-recombinant bovine somatotropin (rbST) antibodies in milk extracts was developed. RbST administration to cows increases their milk production, but is illegal in the EU and a public health concern in the USA. The cellphone monitors the presence of anti-rbST antibodies (rbST biomarker), which are endogenously produced upon administration of rbST and excreted in milk. The rbST biomarker present in milk extracts was captured by rbST covalently coupled to paramagnetic microspheres and labeled by quantum dot (QD)-coupled detection antibodies. The emitted fluorescence light from these captured QDs was then imaged using the cellphone camera. Additionally, a dark-field image was taken in which all microspheres present were visible. The fluorescence and dark-field microimages were analyzed using a custom-developed Android application running on the same cellphone. With this setup, the microsphere fluorescence immunoassay and cellphone-based detection were successfully applied to milk sample extracts from rbST-treated and untreated cows. An 80 % true-positive rate and 95 % true-negative rate were achieved using this setup. Next, the cellphone-based detection platform was benchmarked against a newly developed planar imaging array alternative and found to be equally performing versus the much more sophisticated alternative. Using cellphone-based on-site analysis in future residue monitoring can limit the number of samples for laboratory analysis already at an early stage. Therewith, the entire monitoring process can become much more efficient and economical. Figure
Cellphone-based detection platform for rbST biomarker analysis in milk extracts using a microsphere fluorescence immunoassay相似文献
TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors). 相似文献
We developed a polymeric 2-DE chip system. The chip consisted of an IEF region, an SDS-PAGE region, a valveless connection port, and a sample introduction port. A "junction structure" as a valveless connection port, which allowed separating and connecting the first- and second-dimensional gels, was fabricated between their regions. A "solution inlet" as a sample introduction port was fabricated to perform the liquid and sample introductions without solution leakage. Simultaneous sample monitoring was performed using the on-chip detection system. The performances of the system were demonstrated using commercially available proteins as a standard specimen and tissue-extracted proteins as the real samples. All procedures were employed without any movement of relocation part. This new 2-D separation system realized improved labor-intensive operations and a reduced experimental time. 相似文献