A High-Throughput Single-Cell Assay on a Valve-Based Microfluidic Platform Applied to Protein Quantification, Immune Response Monitoring, and Drug Discovery.

The use of microfluidic technology in single-cell assay has shown potential in biomedical applications like protein quantification, immune response monitoring, and drug discovery. Because of the details of information that can be obtained at single-cell resolution, the single-cell assay has been applied to tackle challenging issues such as cancer treatment. Information like the levels of protein expression, cellular heterogeneity, and unique behaviors within subsets are very important in the biomedical field. For a single-cell assay system, a high-throughput platform that can do on-demand media exchange and real-time monitoring is advantageous in single-cell screening and profiling. In this work, a high-throughput valve-based device is presented, its use in single-cell assay, particularly in protein quantification and surface-marker analysis, and its potential application to immune response monitoring and drug discovery are laid down in detail.

Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns.

A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

The future of microfluidics in immune checkpoint blockade.

Recent advances in microfluidic techniques have enabled researchers to study sensitivities to immune checkpoint therapy, to determine patients' response to particular antibody treatment. Utilization of this technology is helpful in antibody discovery and in the design of personalized medicine. A variety of microfluidic approaches can provide several functions in processes such as immunologic, genomic, and/or transcriptomic analysis with the aim of improving the efficacy and coverage of immunotherapy, particularly immune checkpoint blockade (ICB). To achieve this requires researchers to overcome the challenges in the current state of the technology. This review looks into the advancements in microfluidic technologies applied to researches on immune checkpoint blockade treatment and its potential shift from proof-of-principle stage to clinical application.

In-vitro study of monocytic THP-1 leukemia cell membrane elasticity with a single-cell microfluidic-assisted optical trapping system.

We studied the elastic profile of monocytic THP-1 leukemia cells using a microfluidic-assisted optical trap. A 2-µm fused silica bead was optically trapped to mechanically dent an immobilized single THP-1 monocyte sieved on a 15-µm microfluidic capture chamber. Cells treated with Zeocin and untreated cells underwent RT-qPCR analysis to determine cell apoptosis through gene expression in relation to each cell's deformation profile. Results showed that untreated cells with 43.05 ± 6.68 Pa are more elastic compared to the treated cells with 15.81 ± 2.94 Pa. THP-1 monocyte's elastic modulus is indicative of cell apoptosis shown by upregulated genes after Zeocin treatment. This study clearly showed that the developed technique can be used to distinguish between cells undergoing apoptosis and cells not undergoing apoptosis and which may apply to the study of other cells and other cell states as well.

A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling.

Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.

Accurate detection of carcinoma cells by use of a cell microarray chip.

BACKGROUND: Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment. METHODS AND FINDINGS: A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM) cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM) monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%), accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies. CONCLUSION: The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level.

Discrimination of primitive endoderm in embryoid bodies by Raman microspectroscopy.

Embryoid bodies (EBs), derived from aggregated embryonic stem (ES) cells, are capable of differentiating into all three germ layers, including the endoderm, mesoderm, and ectoderm. The initial stage of EB differentiation is the formation of a primitive endoderm (PE) layer located at the periphery of the aggregate. Raman microspectroscopy was employed to segregate PE cells from undifferentiated ES cells. The Raman spectra of the PE cells of the periphery of EBs, formed upon the withdrawal of leukemia inhibitory factor (LIF), were compared with those of the undifferentiated ES cells of the core of cell aggregates, formed in the presence of LIF. It was noticed that the PE cells have high contents of proteins and low contents of nucleic acids, lipids, and carbohydrates compared with ES cells. Also, we established the presence of another population of PE cells located in the core of the EBs. In addition, we identified some specific Raman markers to distinguish PE cells from ES cells (e.g., I(1003)/I(937)). This is the first study to investigate the PE cells of live EBs and define some Raman markers to distinguish them from undifferentiated ES cells.

A rapid sample pretreatment protocol: improved sensitivity in the detection of a low-abundant serum biomarker for prostate cancer.

We have developed a rapid immunoglobulin G (IgG) and a human serum albumin (HSA) depletion protocol. We depleted both HSA and IgG (> 97%) separately, and in a single procedure. The method is specific and reproducible (RSD < 1.0%), and substantially lowered the detection limit of prostate-specific antigen, a prostate cancer biomarker. The method can be applied to other biomarkers and proteomic studies. Interestingly, the depletion of HSA might not be blankly as beneficial as widely portrayed. Our study suggests the depletion of IgG to be more beneficial than albumin depletion.

Label-free electrical sensing of small-molecule inhibition on tyrosine phosphorylation.

Protein tyrosine kinases (PTKs) play a central role in human carcinogenesis and have emerged as the promising new targets. Small-molecule inhibitors of PTKs have shown impressive anticancer effects and are rapidly entering the clinic. PTK assays allow for high-throughput identification of small-molecule inhibitors. However, current methods of detecting kinase activity require the use of radioisotopes or expensive reagents; such as fluorescently labeled antibodies. We have developed a novel label-free approach for the quantitative detection of peptide tyrosine (Tyr) phosphorylation using the electrochemical oxidation current signal of Tyr. When the phosphorylation is achieved, the phosphorylated Tyr (Tyr-P) cannot be oxidized at approximately 0.65 V. However, when the phosphorylation is successfully inhibited using a small molecule, Tyr can be oxidized and result in a high current response on a multiwalled carbon nanotube-modified screen-printed carbon electrode. We determined the activity of cellular-sarcoma (c-Src) nonreceptor PTK, p60(c-Src), in combination with its highly specific substrate peptide, Raytide. Tyr kinase reactions were also performed in the presence of a well-defined small-molecule inhibitor, 4-amino-5-(4-chlorophenyl)-7- (tert-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Based on the dependency of Tyr oxidation signal on inhibitor concentration, IC50 value, half-maximal inhibition of the inhibitor, was estimated as 5 nM for PP2. Our label-free electrochemical method is a promising candidate for pharmaceutical research and development in screening small-molecule inhibitors of PTKs.

Gold nanoparticle-based electrochemical detection of protein phosphorylation.

In this report, we demonstrate the application of Au nanoparticles in the electrochemical detection of protein phosphorylation. The method is based on the labeling of a specific phosphorylation event with Au nanoparticles, followed by electrochemical detection. The phosphorylation reaction is coupled with the biotinylation of the kinase substrate using a biotin-modified adenosine 5'-triphosphate [gamma]-biotinyl-3,6,9-trioxaundecanediamine (ATP) as the co-substrate. When the phosphorylated and biotinylated kinase substrate is exposed to streptavidin-coated Au nanoparticles, the high affinity between the streptavidin and biotin resulted in the attachment of Au nanoparticles on the kinase substrate. The electrochemical response obtained from Au nanoparticles enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. We determined the activity of Src non-receptor protein tyrosine kinase, p60(c-Src) and protein kinase A in combination with their highly specific substrate peptides Raytide EL and Kemptide, respectively. The detection limits for Raytide EL and Kemptide were determined as 5 and 10 microM, (S/N=3), and the detection limits for the kinase activity of p60(c-Src) and protein kinase A (PKA) were determined as 5 and 10 U mL(-1), (S/N=3), respectively. Tyrosine kinase reactions were also performed in the presence of a well-defined inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP2), and its negative control molecule, 4-amino-7-phenylpyrazol[3,4-d] pyrimidine (PP3), which had no inhibition effect. Based on the dependency of Au nanoparticle signal on inhibitor concentration, IC(50) value, half-maximal inhibition of the inhibitors was estimated. IC(50) values of PP2, genistein and herbimycin A to p60(c-Src) were detected as 5 nM, 25 microM and 900 nM, respectively. The inhibition of PKA activity on Kemptide using ellagic acid was monitored with an IC(50) of 3.5 microM. The performance of the biosensor was optimized including the kinase reaction, incubation with streptavidin-coated Au nanoparticles, and the small molecule inhibitors. Kinase peptide-modified electrochemical biosensors are promising candidates for cost-effective kinase activity and inhibitor screening assays.

Label-free immunosensor for prostate-specific antigen based on single-walled carbon nanotube array-modified microelectrodes.

We have fabricated a label-free electrochemical immunosensor using microelectrode arrays modified with single-walled carbon nanotubes (SWNTs). Label-free detection of a cancer marker, total prostate-specific antigen (T-PSA), was carried out using differential pulse voltammetry (DPV). The current signals, derived from the oxidation of tyrosine (Tyr), and tryptophan (Trp) residues, increased with the interaction between T-PSA on T-PSA-mAb covalently immobilized on SWNTs. The selectivity of our biosensor was challenged using bovine serum albumin (BSA) as the target protein. The detection limit for T-PSA was determined as 0.25 ng/mL. Since the cut-off limit of T-PSA between prostate hyperplasia and cancer is 4 ng/mL, the performance of our label-free electrochemical immunosensor seems promising for further clinical applications.

Quantum dot-based immunosensor for the detection of prostate-specific antigen using fluorescence microscopy.

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.

Selection and properties for the recognition of P19 embryonic carcinoma stem cells.

The P19 cell is a pluripotent stem cell of murine teratocarcinoma. When treated with retinoic acid, P19 cells can be differentiated along a neural cell lineage in culture. To isolate peptides that bind to the stem cell, we employed a phage display technology with undifferentiated P19 cells as the target. To reduce nonspecific binding of phages to the cell surface, the phage libraries were preadsorbed to the differentiated P19 cells before each selection on the undifferentiated P19 cells. After eight rounds of the selection, No. 28 phage displaying ALPSTSSQMPQL-peptide was isolated. Immunofluorescence analysis revealed that No. 28 phage selectively binds to the undifferentiated P19 cells but not to the differentiated P19 cells or SHSY cell line. The chemically synthesized peptide ALPSTSSQMPQL presented on the No. 28 phage efficiently inhibited the binding of No. 28 phage to the undifferentiated P19 cells. This result confirmed that No. 28 phage binding to the cell was mediated by the displayed peptide. The identified peptide may be targeted to a marker expressed on the stem cell and thus become a practical tool for the isolation of somatic stem cells.

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles.

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.

Resin-based micropipette tip for immunochromatographic assays in urine samples.

A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 microl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 microl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity.

Damage to and recovery of coastlines polluted with C-heavy oil spilled from the Nakhodka.

The damage to and recovery of the Japanese coastline from Suzu, Ishikawa Prefecture to Mikuni, Fukui Prefecture was investigated visually over three years after a C-heavy oil spill from the Russian tanker "Nakhodka" in the Japan Sea on January 2, 1997. The beached C-heavy oil tended to remain for a long time on coasts of bedrock and boulder/cobble/pebble but it was removed rapidly from coasts of gravel/sand and man-made structures such as concrete tetrapods. On the coasts of the latter type, wave energy appeared to be the main force removing the oil. One year after the spill, C-heavy oil tended to remain strongly on the sheltered coasts of bedrock and boulder/cobble/pebble. Even on coasts of this type, the contamination was remarkably absent by 2 years after the spill. The concentration levels of polycyclic aromatic hydrocarbons (PAHs) in oil lumps, sand and seawater were monitored during 3 years following the spill. The concentrations of PAHs having 2 or 3 rings decreased more quickly than did those of PAHs having 4 or more rings, suggesting that volatilization was the main cause of the decrease. On the other hand, the concentrations of PAHs having 4 to 6 rings did not start to decrease until 7 months after the spill. The main cause of the decrease seemed to be photolysis. The concentration of BaP in seawater off the polluted coasts was high 1 month after the spill and then decreased. Three years after the spill, the level fell to the sub ng/L level, which was as low as the level in seawater along unpolluted clean coasts in Japan. The concentration of BaP in greenling was higher than the normal level only during the first two months after the spill. These results suggest that the coastlines in Ishikawa and Fukui Prefectures that were polluted with C-heavy oil recovered in 3 years.

Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor.

The development of label-free optical biosensors for DNA and other biomolecules has the potential to impact life sciences as well as screening in medical and environmental applications. In this report, we developed a localized surface plasmon resonance (LSPR) based label-free optical biosensor based on a gold-capped nanoparticle layer substrate immobilized with peptide nucleic acids (PNAs). PNA probe was designed to recognize the target DNA related to tumor necrosis factor. The nanoparticle layer was formed on a gold-deposited glass substrate by the surface modified silica nanoparticles using silane-coupling reagent. The optical properties of gold-capped nanoparticle layer substrate were characterized through monitoring the changes in the absorbance strength, as the thickness of the biomolecular layer increased with hybridization. The detection of PNA-DNA hybridization with target oligonucleotides and PCR-amplified real samples were performed with a limit of detection value of 0.677 pM target DNA. Selective discrimination against a single-base mismatch was also achieved. Our LSPR-based biosensor with the gold-capped nanoparticle layer substrate is applicable to the design of biosensors for monitoring of the interaction of other biomolecules, such as proteins, whole cells, or receptors with a massively parallel detection capability in a highly miniaturized package.

Electrochemical coding of single-nucleotide polymorphisms by monobase-modified gold nanoparticles.

Rapidly increasing information about the human genome requires a fast and simple method for the detection of single-nucleotide polymorphisms (SNPs). To date, the conventional SNP detection technologies have been unable to identify all possible SNPs and needed further development in cost, speed, and sensitivity. Here we describe a novel method to discriminate and code all possible combinations. SNPs were coded by monitoring the changes in the electrochemical signal of the monobase-modified colloidal gold (Au) nanoparticles. First, a chitosan layer was formed on the alkanethiol self-assembled monolayer-modified Au nanoparticle. The monobases were then attached onto the chitosan-coated Au nanoparticles through their 5' phosphate group via the formation of a phosphoramidate bond with the free amino groups of chitosan. The size of the surface-modified Au nanoparticle was found to be 8.46 +/- 1.53 nm by using atomic force microscopy. If there is a SNP in DNA and the mismatched bases are complementary to the monobase, Au nanoparticles accumulate on the electrode surface in the presence of DNA polymerase I (Klenow fragment), thus resulting in a significant change in the Au oxide wave. In this report, monobase-modified Au nanoparticles show not only the presence of a SNP, but also identify which bases are involved within the pair. Especially, the identification of a transversion SNP, which contains a couple of the same pyrimidine or purine bases, is greatly simplified. A model study was performed by using a synthetic 21-base DNA probe related to tumor necrosis factor (TNF-alpha) along with its all possible mutant combinations. This versatile nanoparticle-based electrochemical protocol is a promising candidate for coding all mutational changes.

Application of on-chip cell cultures for the detection of allergic response.

In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3), a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope.

Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.