Xuefei Lv

Microfluidics for cell-cell interactions: A review

Microfluidic chip has been applied in various biological fields owing to its low-consumption of reagents, high throughput, fluidic controllability and integrity. The well-designed microscale intermediary is also ideal for the study of cell biology. Particularly, microfluidic chip is helpful for better understanding cell-cell interactions. A general survey of recent publications would help to generalize the designs of the co-culture chips with different features. With ingenious and combinational utilization, the chips facilitate the implementation of some special coculture models that are highly concerned in a different spatial and temporal way.

Whole-cell based aptamer selection for selective capture of microorganisms using microfluidic devices

Selective capture of microorganisms is an effective and rapid technique for microbe detection. In this article, we obtained aptamers targeting live bacterial cells E. coli as recognition and capture elements by using a whole-cell based aptamer selection, and integrated the isolated aptamers into a microfluidic device for selective capture of microorganisms. Eight rounds of selection were performed; the specificity and affinity of aptamers to the target bacterial cells were also characterized using flow cytometric analysis. A total of 40 aptamer sequences were obtained, and the apparent dissociation constant Kd of the representative aptamer displaying the highest binding affinity was estimated to be 24.8 ± 2.7 nM. After sequencing of the pool showing the highest binding affinity to E. coli, a DNA sequence of high affinity to the bacteria was integrated into the microfluidic device as recognition and capture elements for selective capture of microorganisms. The results demonstrated that the aptamer-based microfluidic device was able to capture E. coli selectively to distinguish different species of bacteria. This investigation is envisaged to provide a new avenue for the development of lab-on-chip devices for selective capture of microorganisms, which could be beneficial for portable, rapid and economic microbial detection in food safety, environmental monitoring and medical diagnosis.

Microfluidic chip with thermoresponsive boronate affinity for the capture-release of cis-diol biomolecules.

The polydimethylsiloxane microfluidic chip grafted with poly(N-isopropylacrylamide-3-acrylamidephenylboronic acid) (P(NIPAAm-co-AAPBA)) was fabricated by UV-induced grafting polymerization for the capture-release of cis-diol-containing biomolecules by temperature-modulated changes instead of changing the pH value of the mobile phase. Based on the optimal time for benzophenone soaking and UV irradiation of grafting polymerization, P(NIPAAm-co-AAPBA) was successfully grafted on the polydimethylsiloxane substrates, which were characterized by scanning electron microscopy, water contact angle measurements, and Fourier transform infrared spectroscopy. The P(NIPAAm-co-AAPBA)-grafted polydimethylsiloxane microfluidic chip can be successfully used for the capture and release of cis-diol-containing adenosine by adjusting the temperature from 4 to 55°C, and the result was validated by Triple Quad liquid chromatography with mass spectrometry. With further development, the fabricated polydimethylsiloxane microfluidic chips might be chosen as a potential tool for the capture and release of cis-diol-containing macromolecules, such as horseradish peroxidase and glycoprotein.

An aptamer-based trypsin reactor for on-line protein digestion with electrospray ionization tandem mass spectrometry

An immobilized trypsin reactor that was based on aptamers has been developed and applied for the first time for proteomic digestion. Briefly, 25 single-stranded DNA aptamers that were specific for trypsin were obtained after SELEX (systematic evolution of ligands by exponential enrichment) selection. TApt.23 (no. 23 trypsin aptamer), which had the lowest dissociation constant (Kd) value (0.0123 μM), was amino-modified and subsequently grafted to an amino-modified silica surface with glutaraldehyde. The results indicated that 14.65±0.35 μg of trypsin could be immobilized on 10mg of TApt.23-silica when an optimized borate buffer was used. Subsequently, a trypsin reactor was fabricated by using a PEEKsil micro column. Compared with in-solution digestion, the aptamer-based trypsin reactor exhibited similar results for protein identification but used a much shorter digestion time (∼30 min). An on-line analysis platform, which included a trypsin reactor coupled to a high-performance liquid chromatography tandem mass spectrometry device, was built through a 6-port valve and achieved efficient protein digestion compared with in-solution and off-line methods. Compared with irreversible covalent enzyme immobilization, the aptamer-based carrier enables more rapid and convenient immobilized trypsin elution as well as re-immobilization of the enzyme. This superior reactor demonstrated that an aptamer could become a more widely used method for enzyme immobilization and other applications.

Rapid and Efficient Proteolysis for Protein Analysis by an Aptamer-Based Immobilized Chymotrypsin Microreactor

Efficient protein digestion is a key step for successful mass spectrometry identification. However, traditional in-solution digestion suffers some drawbacks, such as autolysis of protease, long analysis times and lack of control. Recently, specific single-stranded nucleic acids, aptamers, screened from random sequence pools, have been performed high affinity for targets. In this paper, we have developed a novel enzyme reactor, which immobilized chymotrypsin based on aptamer-grafted silica beads. Mixed proteins, which consist of bovine serum albumin, myoglobin, and cytochrome c, were used as samples, to evaluate the digestion performance of the enzymatic reactor. With the use of this novel tool, proteins were digested in 40 min to an extent similar to that achieved with soluble enzyme at 37°C after 16 h. Moreover, enzymatic reactor regeneration was carried out through chymotrypsin elution and re-immobilization. The advanced characteristics of the aptamer-based chymotrypsin reactor demonstrated that aptamers could serve as novel materials for rapid and efficient enzyme immobilization and application in protein studies.

Immobilization of Chymotrypsin on Silica Beads Based on High Affinity and Specificity Aptamer and Its Applications

Aptamers with high affinity and specificity to targets, bring new approaches to immobilizing proteins or enzymes. In this work, a group of single-stranded DNA aptamers specific for chymotrypsin were obtained by SELEX method in vitro. After investigation and characterization of all aptamers, AptC.1 (abbreviation for the aptamer with the highest affinity for chymotrypsin) was selected and grafted onto silica matrix with the help of glutaraldehyde as linker, and used subsequently to immobilize chymotrypsin. Specifically, it is shown in experiment that, 12.65 µg of chymotrypsin could be immobilized on 10 mg of AptC.1-Silica in 10 mM pH 8.0 borate solutions, and the activity of immobilized enzyme was not inhibited. Bovine serum albumin, myoglobin and cytochrome c were introduced to investigate the enzymatic performance of prepared immobilized chymotrypsin reactor. All these results demonstrated that aptamer could serve as a potential medium for the immobilization of proteins or enzymes.

A BSA coated NOA81 PCR chip for gene amplification

Norland Optical Adhesive 81 (NOA81) is an excellent material for a microfluidic chip, but this chip has been rarely used in the field of gene amplification due to the inhibition effect on the polymerase chain reaction (PCR). In this study, a NOA81 PCR chip (NP-chip) was developed with a simple, fast fabrication method. To overcome this inhibition effect, a simple bovine serum albumin (BSA) coating method was introduced and the BSA coated pH and concentration were optimized for a better gene amplification effect. The PCR results of HLA-DRB1 indicated that the BSA coating method could greatly improve the PCR efficiency on the NP-chip. Moreover, two kinds of PCRs were performed on the NP-chip to validate the effectiveness of the BSA coating method. (i) Gene mutation of anti EB variable region gene (AEB-HC6-6) was observed under the conditions of cobalt radiation. (ii) Short Tandem Repeats (STRs) PCR was performed on the NP-chip as the multiple-primers PCR. The results showed that the NP-chip with BSA coating was able to successfully realize single and multiple primer gene amplification. With further improvement of the precise temperature control, this kind of NP-chip would be widely applied in gene amplification and promote the development of the miniature gene amplification device in point of care testing (PCOT), forensic detection, etc.

Proteoliposome-Based Capillary Electrophoresis for Screening Membrane Protein Inhibitors

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor.

Microvalve controlled multi-functional microfluidic chip for divisional cell co-culture

Pneumatic micro-valve controlled microfluidic chip provides precise fluidic control for cell manipulation. In this paper, a multi-functional microfluidic chip was designed for three separate experiments: 1. Different cell lines were dispensed and cultured; 2. Three transfected SH-SY5Y cells were introduced and treated with methyl-phenyl-pyridinium (MPP+) as drug delivery mode; 3. Specific protection and interaction were observed among cell co-culture after nerve damage. The outcomes revealed the potential and practicability of our entire multi-functional pneumatic chip system on different cell biology applications.

Fluorescence Resonance Energy Transfer Based Biosensor for Rapid and Sensitive Gene-Specific Determination

Integration of nanomaterial and biomolecular recognition is a promising direction in nanotechnology to develop novel molecular analytical and diagnostic techniques. This work reports a biosensor for rapid and sensitive gene-specific detection based on fluorescence resonance energy transfer (FRET). The gene specific detection was employed interactions between graphene oxide and DNA molecules. Due to noncovalent bonding of single stranded DNA with graphene oxide, the fluorescence of the dye on the molecular probe was quenched with high efficiency; however, in the presence of the specific target, the fluorescence of the biosensing system recovered with a high signal-to-background ratio. The nanobiosensor exhibited high sensitivity and excellent specificity in the determination of a gene for hepatitis B virus MD26. The polymeric material polyvinylpyrrolidone was shown to enhance the limit of detection for specific genes. With the advantages of simple, rapid, sensitive, and inexpensive determination, the graphene oxide based nanobiosensor is a promising tool for molecular recognition in clinical diagnosis.