Immuno-PCR: An ultrasensitive immunoassay for biomolecular detection

Techniques that combine nucleic acid amplification with an antibody-based assay can dramatically increase the sensitivity of conventional immunoassays. This review summarizes the methodology and applications of one such protein detection technique that has been used for the past 23 years—the immuno-polymerase chain reaction (usually referred to as immuno-PCR or IPCR). The key component of an immuno-PCR is a DNA–antibody conjugate that serves as a bridge to link the solid-phase immunoreaction with nucleic acid amplification. The efficiency of immuno-PCR enables a 10- to 10⁹-fold increase in detection sensitivity compared with that of ELISA. Advancements in immuno-PCR have included improvements of production of the DNA–antibody conjugate, assay formats, and readout methods. As an ultrasensitive protein assay, immuno-PCR has a broad range of applications in immunological research and clinical diagnostics.     

Thermal lens detection in microfluidic chips

Main possibilities of thermal lens microscopy, a highly sensitive method of molecular absorption spectroscopy, in chemical analysis in microfluidic chips are shown.     

Immobilization and detection of DNA on microfluidic chips

With the rapid development of micro-Total Analysis Systems (muTAS) and sensitive DNA recognition technologies, it is possible to immobilize DNA probes to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micron-sized, spatially segregated DNA recognition elements in Polydimethylsiloxane (PDMS) microfluidic structures. UV light was used to initiate attachment of a photoactive biotin molecule to the substrate surface. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or non-labeled avidin and biotinylated DNA probes. These techniques were applied to create a prototype microfluidic sensor device that was used to separate and identify synthetic DNA targets that were fluorescently-labeled.     

A capacitive immunosensor for detection of cholera toxin

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹⁰ M under optimized conditions. The limit of detection (LOD) was 1.0 × 10⁻¹⁴ M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2 × 10⁻¹² M and a working range from 3.7 × 10⁻¹¹ to 2.9 × 10⁻¹⁰ M whereas, the later had an LOD of 1.0 × 10⁻¹¹ M and a linearity ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant (K_A) of 1.4 × 10⁹ M⁻¹ was estimated.     

Detection of cholera toxin in seafood using a ganglioside-liposome immunoassay

Microbiological contamination of foods continues to be a major concern in public health. Biological toxins are one class of important contaminants that can cause various human diseases. Outbreaks related to contamination by biological toxins or toxin-producing microorganisms have made it extremely important to develop rapid (approximately 20 min), sensitive and cost-effective analytical methods. This paper describes the development of a sensitive bioassay for the detection of cholera toxin (CT) in selected seafood samples, using ganglioside-incorporated liposomes. In this study, the assays were run with food samples spiked with various concentrations of CT. The limit of detection (LOD) increased by a factor of about 10–20 in most food samples, compared with the LOD in the buffer system previously reported. However, the LOD of toxins in food samples (8 × 10–3 × 10³ fg/mL for CT) was still comparable to, or lower than, that previously reported for other assays. The results from this study demonstrate that the bioassays using ganglioside-liposomes can detect the toxin directly in the field screening of food samples rapidly, simply and reliably, without the need for complex instrumentation.     

Recent advances in surface-enhanced Raman scattering detection technology for microfluidic chips

Microfluidic chip devices and their application to sensitive chemical and biological analyses have attracted significant attention over the past decade. The miniaturization of reaction systems offers practical advantages over conventional benchtop systems. In this case, however, a highly sensitive on-chip detection method is important for the monitoring of chemical reactions as well as for the detection of analytes inside the channel because the detection volume in a micrometer-size channel is extremely small. Recently, a surface-enhanced Raman scattering (SERS) technique is being regarded as a potential candidate for the highly sensitive detection of analytes in a microfluidic chip. This review provides a general survey and an in-depth look at recent developments in SERS techniques for the biological/environmental analysis of minute analytes in a microfluidic chip.     

Magnetic beads-based multicolor colorimetric immunoassay for ultrasensitive detection of aflatoxin B1

Aflatoxin B₁ (AFB₁) is one of the most toxic, mutagenic and carcinogenic mycotoxin, widely exists in contaminated food, grains and feedstuff products. In this study, a novel magnetic beads multicolor colorimetric immunoassay (MBMCIA) based on Au@Ag nanorods (Au@Ag NRs) is proposed to visual detect ultralow concentration of AFB₁ with high-resolution by the naked-eye. To design the MBMCIA system, AFB₁-BSA conjugates were first coated on the surface of magnetic beads (MBs), then alkaline phosphatase (ALP) as a bridge between immunoassay and color reaction was used for catalytic hydrolysis of ascorbic acid-phosphate to generate reductive ascorbic acid. Finally, the yielded ascorbic acid could reduce silver ions to grow a silver coating on the surface of gold nanorods to generate Au@Ag NRs, which leads to the bule-shifted longitudinal absorption peak of Au NRs, accompanying with a series of perceptible color change. Under the optimal conditions, the proposed MBMCIA exhibited good sensitivity and specificity for the detection of AFB₁ with the detection limit as low as 5.7 pg/mL. Meanwhile, the MBMCIA was also applied for the analysis of AFB₁ in spiked wheat samples, the obtained recoveries range from 99.1% to 104.3% with relative standard deviation (RSD) less than 7.05% were acceptable. The proposed MBMCIA integrates separated, enriched, anti-interference and signal read-out into one, which opens up a new avenue for an on-site visual food safety inspection or environmental monitoring.     

Lateral flow immunoassay for progesterone detection

A new express method based on lateral flow immunoassay (LFIA) for progesterone detection was developed. To increase the assay sensitivity an enzyme label (horse-radish peroxidase) was used instead of colloidal gold. An optimal assay format was chosen and the influence of a range of buffer supplements (detergents, proteins and sucrose) was investigated by enzyme-linked immunosorbent assay (ELISA). Linear range of LFIA was between 2 and 40 ng/mL in buffer. Limit of detection was 2 ng/mL, assay time was within 15 min.     

微流控纸芯片在环境分析检测中的应用

近年来,微流控纸芯片由于低成本、便携化、检测快等优点,在需要快速检测的环境分析领域中展现出了巨大的应用前景.该综述从微流控纸芯片在环境分析中的应用角度,总结归纳了微流控纸芯片在环境分析中的最新研究进展,并展望了其在未来的发展趋势与挑战.论文内容引用150余篇源于科学引文索引(SCI)与中文核心期刊中的相关论文.该综述包...     

A galactose polyacrylate-based hydrogel scaffold for the detection of cholera toxin and staphylococcal enterotoxin B in a sandwich immunoassay format

A galactoside-based polyacrylate hydrogel was used as a scaffold to immobilize antibodies for the development of a sandwich immunoassay to detect cholera toxin (CT) and staphylococcal enterotoxin B (SEB). The hydrogel possesses large pores and simulates a solution-like environment allowing easy penetration of large biomolecules. Highly crosslinked hydrogels containing pendant amine or carboxyl functionalities were polymerized through a free-radical polymerization process. Covalent crosslinking of the antibodies on hydrogel films was accomplished using a homobifunctional crosslinker or carbodiimide chemistry. Utilizing the two different crosslinking methodologies, our results demonstrated the effectiveness of repetitive additions of crosslinker reactant into a single location on the gel surface. This approach in fact increased the amount of immobilized antibody. Patterned arrays of the immobilized antibodies for sandwich immunoassay development were achieved using a PDMS template containing micro-channels. This template provided a suitable means for applying reagents in multiple cycles. Fluorescence and three-dimensional (3D) imaging by confocal microscopy and laser scanning confocal microscopy of Cy3-labeled anti-CT and/or Cy3-anti-SEB tracer molecules provided qualitative and quantitative measurements on the efficiency of protein immobilization, detection sensitivity and signal-to-noise ratios. As a result of using the galactose polyacrylate-base hydrogel as a platform for immunoassay development, we have successfully been able to achieve low limits of detection for SEB and cholera toxins (1.0 ng mL(-1)). Repetitive additions (>3 cycles) of the crosslinker and antibody have also shown a dramatic increase in the immobilization of antibody resulting in improved immunoassay sensitivity. Fluorescence signal-to-noise ratios using the hydrogel-based immunoassays have been observed as high a 40:1.     

Rapid detection of algal toxins by microfluidic immunoassay

Herein we report fabricating a microfluidic device to monitor harmful algal blooming (HAB). The heterogeneous immuno-enzyme assay was integrated into a self-designed microfluidic chip for rapid and automatic analysis of algal toxins. The device was made from polydimethylsiloxane (PDMS) and was assembled with a home-made control system. The performance of the system was demonstrated by the detection of microcystin, saxitoxin and cylindrospermopsin, the major cyanotoxins. In one single microfluidic chip, multiple samples were controlled and analysed in a parallel manner. Under the optimal conditions, the linear range and the limit of detection of microcystins were 0-5.0 ng mL(-1) and 0.02 ng mL(-1) respectively. The total analysis time was less than 25 min. The designed device was highly automatic, more efficient and economic compared to conventional techniques.     

Portable smartphone-based microfluidic electrochemical immunosensor for ultrasensitive detection of alpha-fetoprotein in human serum

Rapid and accurate tracing of biomarkers is essential for early detecting and diagnosing of cancer. Therefore, a valid and convenient strategy needs to be developed for efficient monitoring of cancer biomarkers. Herein, we constructed a portable microfluidic electrochemical immunosensor based on three-dimensional reduced graphene oxide (3D rGO) doped with gold nanoparticles (Au NPs) for ultrasensitive determination of alpha-fetoprotein (AFP). The designed microfluidic chip, with the advantages of small injection volume, detachable structure and high integration, was fabricated by 3D printing, which only needed 9 μL of reagent to realize the high sensitivity detection. In addition, the 3D Au NPs-rGO composites with high specific surface area and electrons transfer capacity can effectively increase electroactive sites and enhance electrochemical signals. Benefiting from these features, the 3D Au NPs-rGO microfluidic electrochemical immunochip showed a wide detection range between 0.1 pg/mL–200 ng/mL and a best detection limit of 0.045 pg/mL with the high sensitivity of 175.008 μA (ng/mL)⁻¹ cm⁻². Meanwhile, the proposed immunosensor exhibited reliable AFP detection in human serum samples, which demonstrated that this portable smartphone-based microfluidic electrochemical immunosensor hold great promises in clinical detection and huge potential in personalized healthcare.     

Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk

A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M₁ (AFM₁) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM₁ contamination at nanograms per litre level (LOD 20 ng L⁻¹, IC₅₀ 99 ng L⁻¹), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained.     

Electrokinetic flow control in microfluidic chips using a field-effect transistor

A field-effect transistor is developed to control flow in microfluidic chips by modifying the surface charge condition. In this investigation, zeta potential at a particular location is altered locally by applying a gate voltage, while zeta potential at other locations is maintained at its original value. This non-uniform zeta potential results in a secondary electroosmotic flow in the lateral direction, which is used for flow control in microgeometries. Here, microchannel structures and field-effect transistors are formed on polydimethylsiloxane (PDMS) using soft lithography techniques, and a micro particle image velocimetry technique is used to obtain high resolution velocity distribution in the controlled region. The flow control is observed at relatively low gate voltage (less than 50 V), and this local flow control is primarily due to current leakage through the interface between PDMS and glass layers. A leakage capacitance model is introduced to estimate the modified zeta potential for the straight channel case, and excellent agreement is obtained between the predicted and experimental zeta potential results. This leakage-current based field-effect is then applied to a T-channel junction to control flow in the branch channel. Experiments show that the amount of discharge in the branch channel can be controlled by modulating gate voltage.     

A detection system for microfluidic chips based on epifluorescence videomicroscopy and its analytical potentials

An epifluorescence videomicroscopy system with a light-emitting diode as an excitation source and a charge-coupled device matrix as a radiation detector was developed and studied. The system was used for studying mass transfer in microchannels of single- and two-phase systems. It was shown that the developed detection system is suitable for studying mass transfer on a microchip. A one-dimensional physical and mathematical model was developed for mixing processes on a chip. A comparative analysis of experimental results and model concepts demonstrated the diffusion nature of the fluid flow mixing processes. Mass transfer in the two-phase water-octanol system was studied. Dye concentration profiles in a two-phase flow were recorded depending on the time of phase contact. The diffusion nature of mass transfer in the two-phase system was found.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 4, 2005, pp. 357–366.Original Russian Text Copyright © 2005 by Slyadnev, Kazakov, Shcherbaeva, Ganeev, Moskvin, Zolotov.     

Direct-writing colloidal photonic crystal microfluidic chips by inkjet printing for label-free protein detection

Integrating photonic crystals (PC) into microfluidic systems has attracted immense interest for its novel functions. However, it is still a great challenge to fabricate PC microfluidic chips rapidly with complex functions. In this work, a direct-writing colloidal PC microchannel was firstly achieved by inkjet printing and was used for the surface-tension-confined microfluidic immune assay. PC channels with different structure colors have been successfully integrated on one chip. The fabricated chip has the advantages of rapid fabrication, quick fluidic transport and can monitor the fluidic fluxion using the naked eye. Utilizing this PC microfluidic chip, a colorimetric label-free immune assay was realized without nonspecific adsorption interference of the target.     

Cholera toxin subunit B detection in microfluidic devices

Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM₁ was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL⁻¹ for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.     

Polymer microfluidic chips for electrochemical and biochemical analyses

Our recent developments concerning the fabrication of polymer microchips and their applications for biochemical analyses are reviewed. We first describe two methods of fabrication of polymer microfluidic chips, namely UV-laser photoablation and plasma etching that are well suited for the prototyping and mass fabrication of microchannel networks with integrated microelectrodes. These microanalytical systems can be coupled with various detection means including mass spectrometry, and their applications in capillary electrophoresis are presented here. We also present how UV laser photoablation can be used for the patterning of biomolecules on polymer surfaces for generating two-dimensional arrays of microspots to carry out affinity assays. Finally, the use of the microchips for the development of fast affinity and immunological assays with electrochemical detection is presented, demonstrating the potential of these polymer microchips for medical diagnostics and drug discovery.