Lab-on-a-disc for fully integrated multiplex immunoassays

This paper presents a cost-effective, rapid, and fully automated lab-on-a-disc for simultaneous detection of multiple protein biomarkers in raw samples such as whole blood or whole saliva. For the diagnosis of cardiovascular disease, here, a novel centrifugal microfluidic layout was designed to conduct the simultaneous detection of high sensitivity C-reactive protein, cardiac troponin I, and N-terminal pro-B type natriuretic peptide based on a bead-based sandwich type enzyme-linked immunosorbent assay (ELISA). Three reaction chambers are initially interconnected for the common processes such as sample injection, incubation, and washing and then isolated on-demand for the independent processes such as substrate incubation and final detection. The assay performances such as the limit of detection and the dynamic range were comparable with those of the conventional ELISA despite the significant reduction of the minimum sample volume (200 μL), the amount of washing buffer (700 μL), and the total process time (20 min).     

Development of a membrane-based immunofiltration assay for the detection of T-2 toxin

An improved analytical device capable of performing simultaneous immunofiltration-based immunoassay on 30 samples in the presence of reference standards has been developed. The device consists of a rectangular membrane with 36 antibody spotted zones, one end of which was attached to a semirigid polyethylene card. A piece of wetted filter paper between the membrane and the polyethylene card absorbs the added reagent. The assay is a competitive one using T-2 toxin-horseradish peroxidase (T-2 toxin-HRP) as the labeled analyte and 4-chloro-1 naphthol (4CN) as the substrate. Signal amplification was done by the Super-CARD signal amplification method. Semiquantitative results were obtained by visual comparison of the color intensity of a sample spot with those of reference standards. Densitometric analysis was used for quantitation. The method allows rapid and easy determination of T-2 toxin in wheat and poultry feed with detection limits of 12.5 and 25 microg x kg(-)(1), respectively, with accuracy and precision. Matrix interference was eliminated by appropriate dilution of sample extracts with assay buffer. The detection sensitivity in ELISA was 10-fold higher than that in the membrane-based method. Noninfected samples were spiked with T-2 toxin at several concentrations and analyzed by the present method and rapid ELISA. Mean recoveries by both methods were between 80 and 108%. The correlation between the two methods was excellent (R(2) = 0.99).     

ABO, D blood typing and subtyping using plug-based microfluidics

A plug-based microfluidic approach was used to perform multiple agglutination assays in parallel without cross-contamination and using only microliter volumes of blood. To perform agglutination assays on-chip, a microfluidic device was designed to combine aqueous streams of antibody, buffer, and red blood cells (RBCs) to form droplets 30-40 nL in volume surrounded by a fluorinated carrier fluid. Using this approach, proof-of-concept ABO and D (Rh) blood typing and group A subtyping were successfully performed by screening against multiple antigens without cross-contamination. On-chip subtyping distinguished common A1 and A2 RBCs by using a lectin-based dilution assay. This flexible platform was extended to differentiate rare, weakly agglutinating RBCs of A subtypes by analyzing agglutination avidity as a function of shear rate. Quantitative analysis of changes in contrast within plugs revealed subtleties in agglutination kinetics and enabled characterization of agglutination of rare blood subtypes. Finally, this platform was used to detect bacteria, demonstrating the potential usefulness of this assay in detecting sepsis and the potential for applications in agglutination-based viral detection. The speed, control, and minimal sample consumption provided by this technology present an advance for point of care applications, blood typing of newborns, and general blood assays in small model organisms.     

Open-tubular capillary cell affinity chromatography: single and tandem blood cell separation

In this paper, an open-tubular capillary cell affinity chromatography (OT-CAC) method to enrich and separate target cells is described. Open tubular capillaries coated with anti-CD4, anti-CD14, or anti-CD19 antibodies were used as affinity chromatography columns to separate target blood cells. Cells were eluted using either shear force or bubbles. Bubbles were used to elute the captured cells without diluting the captured cells appreciably, while maintaining viability (the viability of the recovered cells was 85.83 +/- 7.34%; the viability of the cells was 90.41 +/- 3.49% before separation). Several aspects of the OT-CAC method were studied, such as the affinity of one antibody between two different cell lines, the effect of shear force, and the recovery of captured cells. Single- and multicell type separations were demonstrated by isolating CD4+ cells with antiCD4 coated capillary and isolating CD4+ and CD19+ cells with two capillaries in tandem from blood samples. In the one cell type isolation test, an average of 87.7% of the recovered cells from antiCD4 capillary were lymphocytes and an average of 97.7% of those lymphocytes were CD4+ cells. In the original blood sample, only 14.2% of the leukocytes were CD4+ cells. Two capillary columns were also run in tandem, separating two blood cell types from a single sample with high purity. The use of different elution shear forces was demonstrated to selectively elute one cell type. This method is an inexpensive, rapid, and effective method to separate target cells from blood samples.     

An analytical device for on-site immunoassay. Demonstration of its applicability in semiquantitative detection of aflatoxin B1 in a batch of samples with ultrahigh sensitivity

A simple analytical device has been developed for performing noninstrumental immunofiltration-based assay on a batch of samples. The device consists of membrane strips, with antibody-immobilized zones, attached to a polyethylene card. A moist filter paper placed between the membrane and the polyethylene card acts as the absorbent body. The device was used to estimate very low concentrations of aflatoxin B1 (AFB1) present in food samples by using an improved catalyzed reporter deposition (Super-CARD) method of signal amplification involving biotinylated tyramine (B-T) and avidin-horseradish peroxidase conjugate. 4-chloro-1-naphthol was used as the substrate for visualization. Semiquantitative results are obtained by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards. Quantitative estimation is possible by densitometric analysis (detection limit 0.25 pg/spot, 0.01 ng mL(-1)). Dilute samples can be assayed by in situ concentration with improved dose-response characteristics. A batch of 12 extracted samples can be analyzed in a single test card within 12 min. Spiked and contaminated samples of groundnut, corn, wheat, cheese, and chilli were analyzed without sample cleanup. The matrix interferences were eliminated by using appropriate dilution of the aqueous methanol extracts. Mean recoveries from different food samples were between 91 and 104%. The values obtained for infected corn and groundnut samples correlated well (R2=0.99) with the estimates by HPLC. The method is well-suited for visual screening of agricultural and food samples for AFB1 under field conditions.     

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40 min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.     

甘油对人的红细胞冷冻干燥保存效果的影响

就甘油对人红细胞冷冻干燥保存效果的影响进行了实验研究。实验中先用不同浓度的甘油对红细胞进行处理，用PVP、柠檬酸钠、蔗糖和胎牛血清作为冻干保护剂对红细胞进行冻干保存，冻干后的样品用质量浓度为30g／L的NaCl复水，用血球分析仪和光镜进行检测。实验结果表明；用甘油进行处理后，红细胞的回收率明显高于没有用甘油处理的红细胞组。其中，采用质量浓度为400g／L的甘油处理后，红细胞冻干回收率达到51．4％。     

Analysis of lipoproteins by capillary zone electrophoresis in microfluidic devices: assay development and surface roughness measurements

The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica capillaries with both UV absorption and laser-induced fluorescence detection. This separation was accomplished using Tricine buffer (pH 9.0) with methylglucamine added as a dynamic coating. With UV detection, LDL eluted as a relatively sharp peak with a migration time of approximately 11 min and HDL eluted as a broad peak with a migration time of 12.5 min. Fluorescence detection of lipoproteins stained with NBD-ceramide was used with the same buffer system to give comparable results. Furthermore, fluorescence staining of human serum samples yielded results similar to the fluorescently stained LDL and HDL fractions, showing that this method can be used to quantify lipoproteins in serum samples. The method was also used to detect lipoproteins in glass micro-CE devices. Very similar results were obtained in microdevices although with much faster analysis times, LDL eluted as a sharp peak at approximately 25 s and HDL as a broad peak at slightly longer time. In addition, higher resolution was obtained on chips. To our knowledge, these results show the first separation and detection of lipoproteins in a microfluidic device using native serum samples. Atomic force microscopy was used to characterize the rms surface roughness (Rq) of microfluidic channels directly. Devices with different surface roughness values were fabricated using two different etchants for Pyrex wafers with a polysilicon masking layer. Using 49% HF, the measured roughness is Rq = 10.9 +/- 1.6 nm and with buffered HF (NH4F + HF) the roughness is Rq = 2.4 +/- 0.7 nm. At this level of surface roughness, there is no observable effect on the performance of the devices for this lipoprotein separation.     

Electrochemical sample matrix elimination for trace-level potentiometric detection with polymeric membrane ion-selective electrodes

Potentiometric sensors are today sufficiently well understood and optimized to reach ultratrace level (subnanomolar) detection limits for numerous ions. In many cases of practical relevance, however, a high electrolyte background hampers the attainable detection limits. A particularly difficult sample matrix for potentiometric detection is seawater, where the high saline concentration forms a major interfering background and reduces the activity of most trace metals by complexation. This paper describes for the first time a hyphenated system for the online electrochemically modulated preconcentration and matrix elimination of trace metals, combined with a downstream potentiometric detection with solid contact polymeric membrane ion-selective microelectrodes. Following the preconcentration at the bismuth-coated electrode, the deposited metals are oxidized and released to a medium favorable to potentiometric detection, in this case calcium nitrate. Matrix interferences arising from the saline sample medium are thus circumvented. This concept is successfully evaluated with cadmium as a model trace element and offers potentiometric detection down to low parts per billion levels in samples containing 0.5 M NaCl background electrolyte.     

Group-type analysis of oxygenated compounds with a silica gel porous layer open tubular column and comprehensive two-dimensional supercritical fluid and gas chromatography

A porous layer open tubular (PLOT) silica gel column was used together with subcritical CO2 as the mobile phase to effect the group separation of polar oxygenated compounds. Aliphatic and aromatic compounds were shown to elute together. This group was followed by ethers and aldehydes, which were separated from compounds containing an alcohol functional group. Compounds with a carboxylic acid moiety could also be eluted from the silica gel. The group separation obtained when a silica gel PLOT column is used together with subcritical CO2 was also demonstrated to be valuable as the first dimension of a comprehensive two-dimensional SFCxGC analysis where the GC analysis in the second dimension is performed with a fast and independently heated temperature programmed gas chromatograph. With this combination of SFC and GC, many of the oxygenated compounds, routinely found in petroleum samples, could successfully be separated and identified.     

Accelerated colorimetric immunosensing using surface-modified porous monoliths and gold nanoparticles

Abstract

A rapid and sensitive immunoassay platform integrating polymerized monoliths and gold nanoparticles (AuNPs) has been developed. The porous monoliths are photopolymerized in situ within a silica capillary and serve as solid support for high-mass transport and high-density capture antibody immobilization to create a shorter diffusion length for antibody–antigen interactions, resulting in a rapid assay and low reagent consumption. AuNPs are modified with detection antibodies and are utilized as signals for colorimetric immunoassays without the need for enzyme, substrate and sophisticated equipment for quantitative measurements. This platform has been verified by performing a human IgG sandwich immunoassay with a detection limit of 0.1 ng ml^?1. In addition, a single assay can be completed in 1 h, which is more efficient than traditional immunoassays that require several hours to complete.     

Serum creatinine detection by a conducting-polymer-based electrochemical sensor to identify allograft dysfunction

Kidney transplant recipients who have abnormally high creatinine levels in their blood often have allograft dysfunction secondary to rejection. Creatinine has become the preferred marker for renal dysfunction and is readily available in hospital clinical settings. We developed a rapid and accurate polymer-based electrochemical point-of-care (POC) assay for creatinine detection from whole blood to identify allograft dysfunction. The creatinine concentrations of 19 blood samples from transplant recipients were measured directly from clinical serum samples by the conducting polymer-based electrochemical (EC) sensor arrays. These measurements were compared to the traditional clinical laboratory assay. The time required for detection was <5 min from sample loading. Sensitivity of the detection was found to be 0.46 mg/dL of creatinine with only 40 μL sample in the creatinine concentration range of 0 mg/dL to 11.33 mg/dL. Signal levels that were detected electrochemically correlated closely with the creatinine blood concentration detected by the UCLA Ronald Reagan Medical Center traditional clinical laboratory assay (correlation coefficient = 0.94). This work is encouraging for the development of a rapid and accurate POC device for measuring creatinine levels in whole blood.     

3D protein microarrays: performing multiplex immunoassays on a single chip

The enzyme-linked immunosorbent assay (ELISA) is typically applied in the format of microtiter plates. To increase throughput and reduce consumption of precious samples, efforts have been made to transfer ELISA to the microchip format using conventional microarrays, microfluidic systems, and chips bearing microwells. However, all three formats lack the possibility to screen several analytes on several immobilized binders at a time or require complicated liquid handling, surface modifications, and additional equipment. Here, we describe an immunoassay performed on a standard microscope slide without the requirement for wells or tubes to separate the samples using standard surfaces and machinery already available for microarray technology. The new multiple spotting technique (MIST) comprises immobilization of a binder onto a surface and subsequent spotting of the second compound on the same spot, on top of the immobilized binder. We show that the analytes bind their ligands immediately within the confined space of separate droplets on the chip surface, thereby eliminating the need for extra incubation time. We illustrate the feasibility of the new technique by spotting dilution rows of proteins or monoclonal and polyclonal antibodies on top of their immobilized binders. Moreover, we demonstrate specificity by applying a mixture of antibodies in a multiplex format and demonstrate that the technique is compatible with conventional microarray protocols, such as total incubation. Finally, we indicate that the technique is capable of quantifying as little as 400 zmol (240,000 molecules) of analyte.     

High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.     

TNT detection using multiplexed liquid array displacement immunoassays

The presence of trace contamination of soil and groundwater with explosives is an ongoing concern, for which improved methods are required to facilitate their detection and quantification. This is true both for the monitoring of remediation and for site characterization. Immunosensors have been found effective for solution-phase detection of environmental contaminants. Our work utilized the Luminex100 (flow cytometer) to detect TNT in a multiplexed displacement immunoassay format. The Luminex100 can perform a multiplexed assay by discriminating between up to 100 different bead sets. We used this capability to evaluate four different TNT monoclonal antibodies, two recombinant TNT antibodies, and a control antibody simultaneously for the rapid detection of TNT and other nitroaromatics. TNT could be detected at 0.1 ppb and quantified over the range of 1.0 ppb to 10 ppm. In addition, the assay was shown to be effective in various matrixes such as lake water, seawater, and acetone extracts of soil. Seawater required dilution with two parts buffer to avoid loss of microspheres, while the acetone extracts were diluted 100-fold or more to minimize solvent affects.     

Macro-/nanoporous silicon as a support for high-performance protein microarrays

The present work demonstrates the possibilities of using macroporous silicon as a substrate for highly sensitive protein chip applications. The formation of 3D porous silicon structures was performed by electrochemical dissolution of monocrystalline silicon. The fabricated macroporous silicon network has a rigid spongelike structure showing high uniformity and mechanical stability. The microfluidic properties of the substrates were found to be essential for a good bioassay performance. Small spot area, good spot reproducibility, and homogeneous spot profiles were demonstrated on the substrates for immobilized aRIgG. Water contact angles were measured on the porous surface and compared to that of planar silicon, silanized glass, and ordinary microscope glass slides. The effect of the porous surface on the performance of a model IgG-binding immunoassay is presented. aRIgG was microdispensed onto the chip surface forming a microarray of spots with high affinity for the target analyte. The dispensing was performed using an in-house-developed piezoelectric flow-through dispenser. Each spot was formed by a single droplet (100 pL) at each position. The macroporous silicon allowed a high-density microarraying with spot densities up to 4400 spots/cm2 in human plasma samples without cross-talk and consumption of only 0.6 pmol of antibodies/1-cm2 array. Antigen levels down to 70 pM were detected.     

基于酵母发酵致孔的小麦麸质蛋白/聚丙烯酸钠复合多孔水凝胶的合成及溶胀性能

以1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐（EDC）活化丙烯酸（AA）羧基与小麦麸质蛋白（WG）接枝交联,酵母菌素分解WG中淀粉等多糖产生的CO₂作为孔模板,水溶液中自由基聚合制备了WG/聚丙烯酸钠（PNaA）多孔复合水凝胶（WG/PNaA）。FTIR分析表明,WG链上-OH、-NH₂等与AA成功接枝,并与中和的丙烯酸钠（NaA）在N,N'-亚甲基双丙烯酰胺（MBA）存在下聚合交联。场发射SEM（FESEM）证实,适量酵母菌素在WG体系中产生的CO₂可作为孔模板,在WG/PNaA网络中形成蜂窝状多孔结构,这种孔状结构不仅提高了WG/PNaA复合水凝胶在蒸馏水和生理盐水中的平衡溶胀倍率,也使其Schott's准二级动力学起始溶胀速率常数K_is提高至无酵母致孔样的5倍,Ritger半经验方程分析也证实其扩散系数n=0.5642,为non-Fickian溶胀,即孔状网络在凝胶溶胀初期有利于水分子快速扩散。考察了WG/PNaA复合水凝胶在蒸馏水-生理盐水、pH为2.2和7.4时磷酸缓冲溶液中的溶胀敏感性。结果表明,经过5次反复溶胀-去溶胀循环后仍具有良好的响应性,即多孔WG/PNaA复合凝胶同时具有灵敏可逆的盐和pH敏感性,为该水凝胶在药物控释领域应用提供了潜在可能。     

Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis II (Hunter Syndrome)

We have developed a tandem mass spectrometry based assay of iduronate-2-sulfatase (IdS) activity for the neonatal detection of mucopolysaccharidosis II (MPS-II, Hunter Syndrome). The assay uses a newly designed synthetic substrate (IdS-S) consisting of α-L-iduronate-2-sulfate, which is glycosidically conjugated to a coumarin and a linker containing a tert-butyloxycarbamido group. A short synthesis of the substrate has been developed that has the potential of being scaled to multigram quantities. Sulfate hydrolysis of IdS-S by IdS found within a 3 mm dried blood spot specifically produces a nonsulfated product (IdS-P) which is detected by electrospray tandem mass spectrometry and quantified using a deuterium-labeled internal standard, both carried out in positive ion mode. Analysis of DBS from 75 random human newborns showed IdS activities in the range of 4.8-16.2 (mean 9.1) μmol/(h L of blood), which were clearly distinguished from the activities measured for 14 MPS-II patients at 0.17-0.52 (mean 0.29) μmol/(h L of blood). The assay shows low blank activity, 0.15 ± 0.03 μmol/(h L of blood). The within-assay coefficient of variation (CV) was 3.1% while the interassay CV was 15%.     

Using a new ligand for solid phase extraction of mercury

The octadecyl silica cartridge as a sorbent and 4-bp db (1,4-bis(4-pyridyl)-2,3-diaza-1,3-butadiene) as a ligand is a simple, rapid and reliable method for extracting and preconcentrating of Hg(II) in real samples prior to cold vapor atomic absorption spectrometry. Sample solutions were passed through a column at pH 4.5 then retained mercury ions on the column were eluted with minimal amount of 0.01 M nitric acid with 3 mL min(-1) flow rate. The effect of pH, type of buffer, flow rate of sample and eluent, type and volume of the eluent were investigated and optimized. At optimum effective parameters, concentration factor and detection limit were achieved 128 and 1.87 ng L(-1), respectively.     

Mass spectrometry after capture and small-volume elution of analyte from a surface plasmon resonance biosensor

The identification of binding partners of proteins by mass spectrometry following specific capture on a biosensor surface is a promising tool for proteomics research and the identification and characterization of protein-protein interactions. Previous approaches include the direct ionization of analyte from the biosensor chip on a matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS) apparatus and the on-chip digestion followed by elution, chromatographic concentration of the fragments, and electrospray mass spectrometry. In the present paper, using the small-volume microfluidic sample manipulation technique with oscillatory flow reported recently (Abrantes et al. Anal. Chem. 2001, 73, 2828-2835), analyte is shown to be eluted from the sensor surface into a small volume of buffer that promotes dissociation from the capture surface and delivery to the mass spectrometer. Both the incubation of the sensor surface with the sample and the recovery of analyte can be achieved with a few microliters and conducted until steady-state is attained. Because the procedure is non-destructive for the sensor surface, multiple cycles of capture and elution allow the transfer and concentration of analyte into the elution buffer. The eluted analyte can be studied directly by MALDI-TOFMS, or subjected to proteolytic digestion for protein identification. Transfer into the elution buffer and MALDI-TOFMS detection was achieved from 5 microL of starting samples containing <50 fmol of analyte. Examples are presented for the specific detection and recovery of a protein from a complex mixture of cytosolic proteins.