Automated sample preparation method for suspension arrays using renewable surface separations with multiplexed flow cytometry fluorescence detection

In this paper, we describe a new method of automated sample preparation for multiplexed biological analysis systems that use flow cytometry fluorescence detection. In this approach, color-encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. This method provides for separation of the biomolecules of interest from other sample matrix components as well as from labeling solutions. After sample preparation, the beads can be released from the renewable surface column and delivered to a flow cytometer for direct on-bead analysis one bead at a time. Using mixtures of color-encoded beads derivatized for various analytes yields suspension arrays for multiplexed analysis. Development of this approach required a new technique for automated capture and release of the color-encoded microspheres within a fluidic system. We developed a method for forming a renewable filter and demonstrate its use for capturing microspheres that are too small to be easily captured in previous flow cells for renewable separation columns. The renewable filter is created by first trapping larger beads in the flow cell, and then smaller beads are captured either within or on top of the bed of larger beads. Both the selective microspheres and filter bed are automatically emplaced and discarded for each sample. A renewable filter created with 19.9 μm beads was used to trap 5.6 μm optically encoded beads with trapping efficiencies of 99%. The larger beads forming the renewable filter did not interfere with the detection of color-encoded 5.6 μm beads by the flow cytometer fluorescence detector. The use of this method was demonstrated with model reactions for a variety of bioanalytical assay types including a one-step capture of a biotinylated label on Lumavidin beads, a two-step sandwich immunoassay, and a one-step DNA binding assay. A preliminary demonstration of multiplexed detection of two analytes using color-encoded beads was also demonstrated. The renewable filter for creating separation columns containing optically encoded beads provides a general platform for coupling renewable surface methods for sample preparation and analyte labeling with flow cytometry detectors for suspension array multiplexed analyses.     

Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus

We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.     

Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems

The present study reports a new three-dimensional (3D) microfluidic platform capable of rapid isolation and detection of cancer cells from a large sample volume (e.g. ~1 mL) by utilizing magnetic microbead-based technologies. Several modules, including a 3D microfluidic incubator for the magnetic beads to capture cancer cells, a microfluidic control module for sample transportation and a nucleic acid amplification module for genetic identification, are integrated into this microsystem. With the incorporation of surface-modified magnetic beads, target cancer cells can be specifically recognized and conjugated onto the surface of the antibody-coated magnetic microbeads by utilizing a swirling effect generated by the new 3D microfluidic incubator, followed by isolating and purifying the magnetic complexes via the incorporation of an external magnet and a microfluidic control module, which washes away any unbound waste solution. Experimental results show that over 90% of the target cancer cells can be isolated from a large volume of bio-samples within 10 min in the 3D microfluidic incubator. In addition, the expressed genes associated with ovarian and lung cancer cells can also be successfully amplified by using the on-chip nucleic acid amplification module. More importantly, the detection limit of the developed system is found to be 5 × 10(1) cells mL(-1) for the target cancer cells, indicating that this proposed microfluidic system may be adapted for clinical use for the early detection of cancer cells. Consequently, the proposed 3D microfluidic system incorporated with immunomagnetic beads may provide a promising automated platform for the rapid isolation and detection of cancer cells with a high sensitivity.     

Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot

Anti-ribonucleoprotein (anti-RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1-68k), a major antigen of anti-RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1-68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1-68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI 7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1-68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti-RNP antibodies.     

DNA-directed protein immobilization on mixed self-assembled monolayers via a streptavidin bridge

The simultaneous detection of multiple analytes is an important consideration for the advancement of biosensor technology. Currently, few sensor systems possess the capability to accurately and precisely detect multiple antigens. This work presents a simple approach for the functionalization of sensor surfaces suitable for multichannel detection. This approach utilizes self-assembled monolayer (SAM) chemistry to create a nonfouling, functional sensor platform based on biotinylated single-stranded DNA immobilized via a streptavidin bridge to a mixed SAM of biotinylated alkanethiol and oligo(ethylene glycol). Nonspecific binding is minimized with the nonfouling background of the sensor surface. A usable protein chip is generated by applying protein-DNA conjugates which are directed to specific sites on the sensor chip surface by utilizing the specificity of DNA hybridization. The described platform is demonstrated in a custom-built surface plasmon resonance biosensor. The detection capabilities of a sensor using this protein array have been characterized using human chorionic gonadotropin (hCG). The platform shows a higher sensitivity in detection of hCG than that observed using biotinylated antibodies. Results also show excellent specificity in protein immobilization to the proper locations in the array. The vast number of possible DNA sequences combine with the selectivity of base-pairing makes this platform an excellent candidate for a sensor capable of multichannel protein detection.     

Development of an ultra-low volume flow cell for surface plasmon resonance detection in a miniaturized capillary electrophoresis system

A miniaturized capillary electrophoresis system coupled to a surface plasmon resonance (SPR) sensor on a microfluidic platform fabricated from PDMS is detailed. A previously described split-flow injection technique is first utilized to manipulate sample into the microfluidic chip, followed by separation within the fused-silica capillary and final off-capillary detection of analytes via SPR. Instead of using commercial SPR flow cells requiring relatively large detection volumes, samples of less than 1 nL volume are utilized. The interface between the CE system and SPR sensor made it possible to detect minute volumes of sample with minimal dispersion. The flow cell has the potential to be applicable to miniaturized flow-injection (FI) systems where submicroliter volumes of sample are frequently only available for analysis. The components present in solution, but not bound to the sensor surface, were also investigated. The sensitivity of the CE-SPR system was similar to that found in UV-spectrometric instruments and nonchromophoric components could also be measured.     

Magnetic domain wall conduits for single cell applications

The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation.     

Magnetic particle based electrochemical sensing platform for PCR amplicon detection

This work reports a novel electrochemical PCR detection platform using magnetic particles as a separation tool. A redox-active intercalator, anthraquinonemonosulfonic acid (AQMS), was firstly intercalated into biotin labeled PCR amplicons, and the resulting complex was then captured by streptavidin-coated magnetic particles (MPs) to form AQMS–DNA–MP conjugates. Subsequently, these conjugates were attracted to the bottom of the tube and separated from the solution by applying an external magnetic field, resulting in a significant reduction of the concentration of solution AQMS. The concentration changes of solution AQMS, which reflect the presence and quantity of PCR amplicons, were monitored by differential pulse voltammetry (DPV) on a chip electrode. PCR cycle number-dependent as well as the initial template DNA concentration-dependent performances were investigated. This electrochemistry based PCR detection platform is simple, convenient and inexpensive, and may have potential applications for practical sample monitoring.     

Dual amplification strategy of highly sensitive thrombin amperometric aptasensor based on chitosan-Au nanocomposites

A highly sensitive and selective electrochemical aptasensor for thrombin was developed. By introducing chitosan-gold nanoparticles and horseradish peroxidase (CS-AuNPs-HRP) conjugates to the sensitive union, the thrombin detection signal was dual amplified. The capture probe was prepared by immobilizing an anti-thrombin aptamer on core-shell Fe(3)O(4)-Au magnetic nanoparticles (AuMNPs) and which was served as magnetic separation material as well. The detection probe was prepared from another anti-thrombin aptamer, horseradish peroxidase (HRP), thiolated CS nanoparticle and gold nanoparticle (CS-AuNPs-HRP-Apt2). In the presence of thrombin, the sandwich structure of AuMNPs-Apt1/thrombin/Apt2-CS-AuNPs-HRP was formed and abundant HRP was captured in it. The resultant conjugates are of magnetic characters and were captured onto the surface of a screen printed carbon electrode (SPCE) to prepare the modified electrode by a magnet located on the outer flank of the SPCE. It was demonstrated that the oxidation of hydroquinone (HQ) with H(2)O(2) was dramatically accelerated by the captured HRP. The electrochemical signal, which correlated to the reduction of BQ (the oxidation product of HQ), was amplified by the catalysis of HRP toward the reaction and the enrichment of HRP on the electrode surface. Under optimized conditions, ultrasensitive and high specific detection for thrombin was realized with the proposed assay strategy. The signal current was linearly correlated to the thrombin concentration in the range of 0.01-10 pM with a detection limit of 5.5 fM (S/N = 3). These results promise extensive applications of this newly proposed signal amplification strategy in protein detection and disease diagnosis.     

Development of a novel stirrerliquid/solid microextraction method for the separation and enrichment of trace levels of active compounds in traditional Chinese medicine

A novel stirrer‐liquid/solid microextraction method was developed for the separation and enrichment of trace levels of curcumin, bisdemethoxycurcumin, and demethoxycurcumin in Rhizoma Curcumae Longae, Radix Curcumae, and Rhizoma Curcumae before their analysis by high‐performance liquid chromatography with ultraviolet detection. In the proposed approach, a magnetic stirrer was immersed in decanol to coat its surface completely with decanol, which was used as an extraction platform. The stirrer coated with decanol is not only a power to agitate the sample solution to constantly update the sample on the stirrer surface but also it can adsorb and extract the target analytes. Some effective parameters, including suitable superficial area of stirrer, extraction solvent, sample phase pH, NaCl concentration, stirring rate, extraction time, sample phase volume, were analyzed and selected. Under the optimal conditions, the linearities are 0.0044–2.20 μg/mL, detection limits are 0.3–0.6 ng/mL, and the extraction content per unit length and enrichment factors of the target analytes are 6.24–9.71/mm and 589–917, respectively. Also, the stirrer‐liquid/solid microextraction mechanism for the extraction and enrichment of the target analytes was analyzed and expounded. The results showed that stirrer‐liquid/solid microextraction is a simple, rapid sample pretreatment approach with a high enrichment factor.     

Pathogen detection in complex samples by quartz crystal microbalance sensor coupled to aptamer functionalized core–shell type magnetic separation

A quartz crystal microbalance sensor (QCM) was developed for sensitive and specific detection of Salmonella enterica serovar typhimurium cells in food samples by integrating a magnetic bead purification system. Although many sensor formats based on bioaffinity agents have been developed for sensitive and specific detection of bacterial cells, the development of robust sensor applications for food samples remained a challenging issue. A viable strategy would be to integrate QCM to a pre-purification system. Here, we report a novel and sensitive high throughput strategy which combines an aptamer-based magnetic separation system for rapid enrichment of target pathogens and a QCM analysis for specific and real-time monitoring. As a proof-of-concept study, the integration of Salmonella binding aptamer immobilized magnetic beads to the aptamer-based QCM system was reported in order to develop a method for selective detection of Salmonella. Since our magnetic separation system can efficiently capture cells in a relatively short processing time (less than 10 min), feeding captured bacteria to a QCM flow cell system showed specific detection of Salmonella cells at 100 CFU mL⁻¹ from model food sample (i.e., milk). Subsequent treatment of the QCM crystal surface with NaOH solution regenerated the aptamer-sensor allowing each crystal to be used several times.     

Ultra-sensitive analysis based on electrochemistry,electrokinetic and magnetic preconcentration of analytes

Some of the challenges with detection of ultra-low concentrations of analytes are to achieve sufficient sensitivity of the measurement and to direct the analyte species to the sensor (electrode) surface. This review describes various strategies that are available to address these challenges: method of electrocatalytic amplification, electrochemical measurements performed in combination with electrokinetic preconcentration of analytes, ultra-sensitive analysis utilizing increased surface area and also the manipulation by the magnetic force.     

Separation method based on affinity reaction between magnetic and nonmagnetic particles for the analysis of particles and biomolecules

A separation method is reported for particle and biochemical analysis based on affinity interactions between particle surfaces under magnetic field. In this method, magnetic particles with immunoglobulin G (IgG) or streptavidin on the surface are flowed through a separation channel to form a deposition matrix for selectively capturing nonmagnetic analytes with protein A or biotin on the surface due to specific antigen (Ag)--antibody (Ab) interactions. This separation method was demonstrated using model reactions of IgG--protein A and streptavidin-biotin on particle surface. The features of this new separation method are (1) the deposited Ag-Ab complex can be examined and further analyzed under the microscope, (2) a kinetic study of complex binding is possible, and (3) the predeposited matrix can be formed selectively and changed easily. The detection limits were about 10(-11) g. The running time was less than 10 min. The selectivities of studied particles were 94% higher than those of label-controlled particles. This method extends the applications of analytical magnetapheresis to nonmagnetic particles. Preliminary study shows that this separation method has a great potential to provide a simple, fast, and selective analysis for particles, blood cells, and immunoassay related applications.     

Label-free biosensing with lipid-functionalized gold nanorods

The fabrication of a label-free mass spectrometry and optical detection-based biosensor platform for the detection of low-abundance lipophilic analytes in complex mixtures is described. The biosensor consists of a lipid layer partially tethered to the surface of a gold nanorod. The effectiveness of the biosensor is demonstrated for the label-free detection of a lipophilic drug in aqueous solution and a lipopeptide in serum.     

多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

Combining biofunctional magnetic nanoparticles and ATP bioluminescence for rapid detection of Escherichia coli

A rapid, specific and sensitive method for assay of Escherichia coli (E. coli) using biofunctional magnetic nanoparticles (BMNPs) in combination with adenosine triphosphate (ATP) bioluminescence was proposed. The BMNPs were fabricated by immobilizing a specific anti-E. coli antibody on the surface of amine-functionalized magnetic nanoparticles (about 20 nm in diameter), and then was applied to capture the target bacteria E. coli from samples. The BMNPs exhibited high capture efficiency to E. coli. Transmission electron microscope (TEM) images showed that the BMNPs were bound to the surface of entire E. coli cells. The target bacteria became magnetic so that could be isolated easily from the sample solution by employing an external magnetic field. The concentration of E. coli captured by the BMNPs was then detected by an ATP bioluminescence method. The optimization of ATP measurement was carried out to improve the detection sensitivity. The proposed method was applied to detect the E. coli inoculated into pasteurized milk with low detection limit (20 cfu/mL) and short detection time (about 1 h).     

Microchip electrophoresis coupled with on-line magnetic separation and chemiluminescence detection for multiplexed immunoassay

A facile and universal strategy for multiplexed immunoassay is proposed. The strategy is based on microchip electrophoresis (MCE) coupled with on-line magnetic separation and chemiluminescence (CL) detection. The system consisted of a microchip, an electromagnet, and a photomultiplier. The realization of multiplexed immunoassay protocol involves sampling magnetic nanoparticles (MNPs) labeled antibodies, N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI) labeled antigens and free antigens in the precolumn reactor, on-line immunoreaction, capturing the MNPs-immunocomplexes, and the separation of unconjugated ABEI-labeled antigens. After on-line magnetic separation, the free ABEI-labeled antigens were transported into the separation channel, and mixed with hydrogen peroxide (H(2) O(2) ) in the presence of horseradish peroxidase in the postcolumn reactor, and producing CL emission. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. As a proof-of-concept, the multiplexed immunoassay was evaluated for the simultaneous determination of five model analytes (i.e. hydrocortisone, corticosterone, digoxin, testosterone, and estriol). The results exhibited excellent precision and sensitivity, the relative standard deviations for nine times detection were lower than 4.7% for all the five components, and the detection limits of five analytes were in the range of 3.6-4.9 nM. The MCE system was validated using two human serum-based control samples containing five analytes.     

Mussel-Inspired Surface-Imprinted Sensors for Potentiometric Label-Free Detection of Biological Species

Using sensors to quantify clinically relevant biological species has emerged as a fascinating research field due to their potential to revolutionize clinical diagnosis and therapeutic monitoring. Taking advantage of the wide utility in clinical analysis and low cost of potentiometric ion sensors, we demonstrate a method to use such ion sensors to quantify bioanalytes without chemical labels. This is achieved by combination of chronopotentiometry with a mussel-inspired surface imprinting technique. The biomimetic sensing method is based on a blocking mechanism by which the recognition reaction between the surface imprinted polymer and a bioanalyte can block the current-induced ion transfer of an indicator ion, thus causing a potential change. The present method offers high sensitivity and excellent selectivity for detection of biological analytes. As models, trypsin and yeast cells can be measured at levels down to 0.03 U mL⁻¹ and 50 CFU mL⁻¹, respectively.     

Mussel‐Inspired Surface‐Imprinted Sensors for Potentiometric Label‐Free Detection of Biological Species

Using sensors to quantify clinically relevant biological species has emerged as a fascinating research field due to their potential to revolutionize clinical diagnosis and therapeutic monitoring. Taking advantage of the wide utility in clinical analysis and low cost of potentiometric ion sensors, we demonstrate a method to use such ion sensors to quantify bioanalytes without chemical labels. This is achieved by combination of chronopotentiometry with a mussel‐inspired surface imprinting technique. The biomimetic sensing method is based on a blocking mechanism by which the recognition reaction between the surface imprinted polymer and a bioanalyte can block the current‐induced ion transfer of an indicator ion, thus causing a potential change. The present method offers high sensitivity and excellent selectivity for detection of biological analytes. As models, trypsin and yeast cells can be measured at levels down to 0.03 U mL⁻¹ and 50 CFU mL⁻¹, respectively.     

基于场放大进样和石墨烯量子点双重富集毛细管电泳分离检测三聚氰胺和双氰胺

通过热解法制备了硫掺杂的石墨烯量子点(S-GQDs),同石墨烯量子点(GQDs)相比,S原子的引入有效改善了GQDs的表面状态和化学特性、增强其对正电荷的捕获能力,使其更易与阳离子相互作用.以S-GQDs为载体,结合电堆积富集技术,发展了一种基于场放大进样(FASI)和S-GQDs放大的双重富集毛细管电泳(CE)分离检...