Isolation and Characterization of Exosomes from Cancer Cells Using Antibody-Functionalized Paddle Screw-Type Devices and Detection of Exosomal miRNA Using Piezoelectric Biosensor.

Exosomes are small extracellular vesicles produced by almost all cell types in the human body, and exosomal microRNAs (miRNAs) are small non-coding RNA molecules that are known to serve as important biomarkers for diseases such as cancer. Given that the upregulation of miR-106b is closely associated with several types of malignancies, the sensitive and accurate detection of miR-106b is important but difficult. In this study, a surface acoustic wave (SAW) biosensor was developed to detect miR-106b isolated from cancer cells based on immunoaffinity separation technique using our unique paddle screw device. Our novel SAW biosensor could detect a miR-106b concentration as low as 0.0034 pM in a linear range from 0.1 pM to 1.0 µM with a correlation coefficient of 0.997. Additionally, we were able to successfully detect miR-106b in total RNA extracted from the exosomes isolated from the MCF-7 cancer cell line, a model system for human breast cancer, with performance comparable to commercial RT-qPCR methods. Therefore, the exosome isolation by the paddle screw method and the miRNA detection using the SAW biosensor has the potential to be used in basic biological research and clinical diagnosis as an alternative to RT-qPCR.

A QCM immunosensor employing signal amplification strategies by enlarging the size of nanoparticles using gold or silver staining.

We present quartz crystal microbalance (QCM) immunosensors for the detection of alpha-fetoprotein (AFP) in a human serum immunoassay with high sensitivity. In this study, we employed three types of signal amplification strategies using size enlargement and/or increase in mass of gold and titanium dioxide nanoparticles. Since the basic principle of the QCM sensor is to measure the change in resonance frequency according to the mass change caused by the molecular interactions on the sensor surface, we were able to quantitatively analyze AFP by sandwich immunoassay using gold or titanium dioxide nanoparticles conjugated with anti-AFP detection antibodies and the subsequent three signal amplification techniques in a similar manner. The signal amplification technologies provide the size expansion of gold nanoparticles by gold or silver staining reaction and mass enhancement by photocatalytic silver staining of titanium dioxide nanoparticles. The limit of detections (LODs) of the AFP immunoassay in human serum by the gold and silver staining-mediated signal amplifications for gold nanoparticles were 56 and 87 pg mL-1, respectively, but by the photocatalytic silver staining signal amplification for titanium dioxide nanoparticles was 118 pg mL-1. This means that the signal amplification method through size enhancement by gold staining of gold nanoparticles further improved the detection ability of the QCM immunosensor.

Sensitivity and reproducibility improvements in a human plasma immunoassay with removal of clotting factors.

Interferences in human plasma immunoassay are severe challenge that affects the sensitivity and reproducibility of the assay. The clotting factor fibrinogen is a negatively charged protein and is one of the most common sources of interference in immunoassays, and its removal increases the sensitivity and reproducibility. Here, we present a highly sensitive and reproducible method for the detection of prostate specific antigen (PSA) in human plasma immunoassays. Protamine sulfate, a highly positively charged protein, was used to precipitate fibrinogen via ionic interaction to improve the sensitivity and reproducibility of human plasma immunoassay. In a sandwich ELISA for PSA using plasma and protamine-treated plasma samples, the limit of detection was improved from 413 pg/mL in plasma to 235 pg/mL in protamine-treated plasma samples, and the coefficient of variation known as a measure of reproducibility was significantly lowered by protamine treatment. The use of protamine sulfate in human plasma immunoassays for detection of PSA using quartz crystal microbalance (QCM) biosensors resulted in increased sensitivity and reproducibility by about 2-fold and 3-fold, respectively, relative to when not using protamine sulfate. Based on these results, protamine sulfate was the best choice to increase the sensitivity and reproducibility in immunoassays using plasma samples.

QCM sensing of miR-21 by formation of microRNA-DNA hybrid duplexes and intercalation on surface-functionalized pyrene.

MicroRNAs (miRNAs) are small non-coding RNA molecules that serve as important biomarkers for a variety of diseases such as cancer and vascular disease. However, sensitive and accurate detection of miR-21 is very challenging in that up-regulation of miR-21 is highly associated with several types of malignant tumors. Here, quartz crystal microbalance (QCM) biosensors were developed for sensitive and specific detection of miR-21 through formation of miR-21-DNA hybrid duplexes and non-specific intercalation of surface-modified pyrene molecules. High selectivity for miR-21 over other miRNAs came from the specific hybridization between miR-21 and gold nanoparticle (AuNP)-conjugated complementary oligonucleotides of miR-21. High sensitivity was obtained through formation of intercalated complexes on the surface with subsequent gold staining signal amplification. Under optimum condition using this strategic approach, our novel QCM biosensors could detect miR-21 concentration as low as 3.6 pM in the entire linear range from 2.5 pM to 2.5 µM with a correlation coefficient of 0.989. In addition, these sensors did not work at all for other miRNAs based on their high selectivity. miR-21 in human brain total RNA and total RNA extracted from A549 cell line could also be successfully detected. Therefore, miRNA detection technology using QCM biosensors and their detection mechanisms have potential as alternatives in biological studies and clinical diagnosis.

Highly sensitive piezoelectric immunosensors employing signal amplification with gold nanoparticles.

We present a quartz crystal microbalance (QCM) immunosensor for highly sensitive detection of prostate-specific antigen (PSA) in a human serum immunoassay. In particular, in this study, we employed signal amplification using and enlarging gold nanoparticles. Because QCM measures the change of resonance frequency according to the mass change occurring on the sensor surface, we could quantitatively analyze PSA based on a tremendous increase in mass by sandwich immunoassay using AuNP-conjugated anti-PSA-detecting antibody enhanced with subsequent gold staining. The limit of detection of the PSA immunoassay in human serum without gold staining enhancement was 687 pg ml-1 but was 48 pg ml-1 with the gold staining-mediated signal amplification. That is, amplifying the signal resulted in increased sensitivity and reproducibility of immunoassay in a human serum.

Efficient and accurate analysis of microRNA using a specific extension sequence.

We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay consists of poly(A) tailing and reverse transcription followed by real-time PCR. In the first step of this reaction, target miRNAs are poly(A) tailed by poly(A) polymerase followed by cDNA synthesis using poly(T) adaptors. In the second step, cDNA is hybridized to the 3'-end of a specific extension sequence that contains part of a miRNA sequence; this cDNA-specific extension sequence hybrid forms the novel PCR template. The PCR template is amplified in a SYBR Green-based quantitative real-time PCR with universal forward and reverse primers. The miR-106b in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative extension-based assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms.

Real-time qRT-PCR assay for the detection of miRNAs using bi-directional extension sequences.

Highly specific detection of miRNAs was performed using a novel bi-directional extension (BDE) assay. After reverse transcription, the cDNA was hybridized to a uniquely designed specific BDE sequence; this cDNA-BDE hybrid forms the PCR template. The PCR template was amplified in a SYBR Green-based quantitative real-time PCR. The miR-145 in human brain total RNA could be detected quantitatively in the range of seven orders of magnitude with high linearity and reproducibility. This innovative BDE assay has several performance advantages over the poly(A) tailing method that include lower CT values, clear gel electrophoresis images, and distinct nucleotide peaks in sequencing chromatograms.

Simultaneous Detection of Exosomal microRNAs Isolated from Cancer Cells Using Surface Acoustic Wave Sensor Array with High Sensitivity and Reproducibility.

We present a surface acoustic wave (SAW) sensor array for microRNA (miRNA) detection that utilizes photocatalytic silver staining on titanium dioxide (TiO2) nanoparticles as a signal enhancement technique for high sensitivity with an internal reference sensor for high reproducibility. A sandwich hybridization was performed on working sensors of the SAW sensor array that could simultaneously capture and detect three miRNAs (miRNA-21, miRNA-106b, and miRNA-155) known to be upregulated in cancer. Sensor responses due to signal amplification varied depending on the concentration of synthetic miRNAs. It was confirmed that normalization (a ratio of working sensor response to reference sensor response) screened out background interferences by manipulating data and minimized non-uniformity in the photocatalytic silver staining step by suppressing disturbances to both working sensor signal and reference sensor signal. Finally, we were able to successfully detect target miRNAs in cancer cell-derived exosomal miRNAs with performance comparable to the detection of synthetic miRNAs.

A trachea-inspired bifurcated microfilter capturing viable circulating tumor cells via altered biophysical properties as measured by atomic force microscopy.

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 µL min(-1), an 8 µm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

A centrifugally actuated point-of-care testing system for the surface acoustic wave immunosensing of cardiac troponin I.

A fully automated point-of-care testing (POCT) system with a surface acoustic wave (SAW) immunosensor was developed for rapid and sensitive detection of cardiac troponin I (cTnI) in body fluid (plasma and whole blood). The assay, based on gold nanoparticle sandwich immunoassay and subsequent gold staining, was performed on the SAW immunosensor packaged inside a disposable microfluidic cartridge. The entire fluidic process, including plasma separation, reagent transport, metering, and mixing, was carried out by controlling the centrifugal force acting on the rotating cartridge and laser-irradiated ferrowax microvalves. On investigation of sensor response to various cTnI concentrations, the system exhibited a high performance with a detection limit of 6.7 pg mL(-1), and the coefficient of variation was less than 10% over the entire test range (10 pg mL(-1) to 25 ng mL(-1)). On comparing this POCT system with a clinically utilized system in a physical laboratory (Centaur® XP; Siemens), a correlation coefficient of 0.998 was found, validating the diagnostic capability of the SAW immunosensor.

Detection of cyanide and sulfide ions by different mechanisms using a fluorescent chemical sensor containing a fluorophore and a potential ligand for metal complexes.

Here, we present a fluorescent chemical sensor for the simultaneous detection of CN- and S2- ions, which are highly toxic to humans and the environment. When the BPMA-Flu-Cu2+ complex comprising BPMA-Flu, a fluorophore combined with Cu2+, was used, the fluorescence was turned off. However, the fluorescence was turned on again by the addition of CN- and S2- ions. BPMA-Flu is a unique compound containing both a fluorophore and a ligand coordinated to a metal ion. This strategy using the proposed BPMA-Flu-Cu2+-based fluorescent chemical sensor facilitated quantitative analysis of CN- and S2- ions with high selectivity and sensitivity, despite varying detection mechanisms. The detection limit was as low as 290 nM and 74 nM for CN- and S2- ions, respectively. The sensor was selective and excluded other anions at concentrations 10-fold higher than those of CN- and S2- ions. The recovery rates did not exceed 10% of the original values for the detection of CN- ions in rainwater and tap water. These results indicate acceptable accuracy and precision for practical applications.

Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood.

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 µm) and DMS-79 small cell lung cancer cells (average diameter, 10 µm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

DNAzyme molecular beacon probes for target-induced signal-amplifying colorimetric detection of nucleic acids.

A novel DNAzyme molecular beacon (DNAzymeMB) strategy was developed for target-induced signal-amplifying colorimetric detection of target nucleic acids. The DNAzymeMB, which exhibits peroxidase activity in its free hairpin structure, was engineered to form a catalytically inactive hybrid through hybridization with a blocker DNA. The presence of target DNA leads to dissociation of the DNAzymeMB from the inactive hybrid through hybridization with the blocker DNA. This process results in recovery of the catalytically active DNAzymeMB, which can catalyze a colorimetric reaction that signals the presence of the target DNA. In addition, a primer was rationally designed to anneal to the blocker DNA of the blocker/target DNA duplex and displace the bound target DNA during the extension reaction. The released target DNA triggers the next cycle involving hybridization with blocker DNA, DNAzymeMB dissociation, primer extension, and target displacement. This unique amplifying strategy leads to the generation of multiple numbers of active DNAzymeMB molecules from a single target molecule and gives a detection limit down to 1 pM, a value that is nearly 3 or 5 orders of magnitude lower than those of previously reported DNAzyme molecular beacon-based DNA detection methods.

Sensitive and simultaneous detection of cardiac markers in human serum using surface acoustic wave immunosensor.

We present a rapid and sensitive surface acoustic wave (SAW) immunosensor that utilizes gold staining as a signal enhancement method. A sandwich immunoassay was performed on sensing area of the SAW sensor, which could specifically capture and detect cardiac markers (cardiac troponin I (cTnI), creatine kinase (CK)-MB, and myoglobin). The analytes in human serum were captured on gold nanoparticles (AuNPs) that were conjugated in advance with detection antibodies. Introduction of these complexes to the capture antibody-immobilized sensor surface resulted in a classic AuNP-based sandwich immunoassay format that has been used for signal amplification. In order to achieve further signal enhancement, a gold staining method was performed, which demonstrated that it is possible to obtain gold staining-mediated signal augmentation on a mass-sensitive device. The sensor response due to gold staining varied as a function of cardiac marker concentration. We also investigated effects of increasing operating frequency on sensor responses. Results showed that detection limit of the SAW sensor could be further improved by increasing the operating frequency.

A visible light-induced photocatalytic silver enhancement reaction for gravimetric biosensors.

We have developed a novel microgravimetric immunosensor using a WO(3) nanoparticle-modified immunoassay and a silver enhancement reaction. When the nanoparticles in silver ion solution (i.e.  AgNO(3)) are exposed to visible light, the silver ions are photocatalytically reduced and form a metallic silver coating on the nanoparticles. This silver coating consequently induces changes in the mass and light absorption spectrum. Although photocatalytic reduction reactions can be achieved using ultraviolet (UV) light and TiO(2) nanoparticles as described in our previous publication (Seo et al 2010 Nanotechnology 21 505502), the use of UV light in biosensing applications has drawbacks in that UV light can damage proteins. In addition, conventional quartz crystal substrates must be passivated to prevent undesirable silver ion reduction on their gold-coated sensing surfaces. We addressed these problems by adopting a visible light-induced photocatalytic silver enhancement method using WO(3) nanoparticles and lateral field excited (LFE) quartz crystals. As a proof-of-concept demonstration of the technique, streptavidin was adsorbed onto an LFE quartz crystal, and its mass was enhanced with biotinylated WO(3) nanoparticles, this being followed by a photocatalytic silver enhancement reaction. The mass change due to the enhancement was found to be > 30 times greater than the mass change obtained with the streptavidin alone.

Purification and characterization of an arginine regulatory protein, ArgR, in Corynebacterium glutamicum.

Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for industrial amino acid production. We previously showed that, in C. glutamicum, argCJBDFRGH arginine biosynthesis genes are clustered but independently transcribed from argC and argG promoters, leading to the generation of two transcripts corresponding to argCJBDFR and argGH. In this report, we show the effect of the C. glutamicum ArgR repressor on argC and argG promoters by overexpressing or disrupting the argR gene. Gel filtration assay results indicate that native ArgR is a hexamer of equal subunits with molecular mass of 110 kDa. Protein sequence analysis revealed the presence of an "SR" (Ser57-Arg58) motif for the DNA binding site at the N-terminal region and the "GTIAGDDTV" motif for arginine binding and its oligomerization at the C-terminal region. An argC or argG promoter-lacZ fusion reporter assay and argR mutational analysis showed that transcription of the argCJBDFR arginine biosynthesis genes is regulated from the argC promoter by ArgR in cooperation with L-arginine in C. glutamicum. This finding was supported by the gel mobility-shift assay showing direct binding of hexameric ArgR to the argC promoter in the presence of L-arginine. Unexpectedly, argGH transcription was not responsive to the level of ArgR repressor and/or arginine. In a further study, a C. glutamicum argR mutant was constructed by disrupting the chromosomal argR gene to manufacture an improved arginine-producing strain. Arginine productivity was increased in the C. glutamicum argR mutant strain under conditions of both limited and excessive arginine.

Quartz crystal microbalance cardiac Troponin I immunosensors employing signal amplification with TiO2 nanoparticle photocatalyst.

A sensitive and highly reproducible cardiac troponin I (cTnI) immunoassay in human serum is a challenging research goal for researchers studying biosensors because cTnI can undergo proteolysis and various modifications in blood. Furthermore, the reproducible detection of cTnI at very low concentrations is also required for diagnosing acute myocardial infarction. Here, we present sensitive and highly reproducible quartz crystal microbalance (QCM) immunosensors for the detection of cTnI in human serum. The unique features of this study are the use of a pair of capture antibodies that bind to different epitopes of cTnI, and the use of a signal amplification technique that enlarged the size of the titanium dioxide nanoparticles using photocatalytic silver staining. Since QCM measures changes in the resonance frequency due to the changes in mass occurring on the sensor surface, it is possible to quantitatively analyze cTnI based on the enormous increase in mass using a sandwich immunoassay and subsequent signal amplification by silver staining. The detection limit of the cTnI immunoassay in human serum without photocatalytic silver staining was 307 pg/ml, but 18 pg/ml in photocatalytic silver staining-mediated signal amplification. Thus, amplifying the signal increased the sensitivity and reproducibility of the cTnI immunoassay in human serum.

Electrochemical cell lysis device for DNA extraction.

We present a novel electrochemical cell lysis device to prepare DNA samples for lab-on-a-chip (LOC) applications. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH(-)) at the cathode as alkaline lytic agents. Cathode and anode chambers are separated by a negatively-charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, to prevent inflow of PCR-amplification inhibitors from the anode chamber, and to minimize binding of DNA molecules. Electric current flow and pH maintenance, which depended on the device design, were two important parameters of the device performance. After optimizing the design and visually confirming cell lysis of Chinese hamster ovary (CHO) cells in a very short amount of time, we directly electrolyzed four bacterial cell types suspended in saline solution. Real-time PCR (qPCR) analysis showed that our device could lyse both gram-positive and gram-negative bacterial cells with higher efficiency than other common methods and could detect DNA on the microlitre scale. Our data demonstrate several advantages of the proposed device: absence of cell lysis chemicals and heating; no adverse effects on PCR amplification; low DNA loss; low voltage and power consumption; and rapid processing. The device could potentially be applied as an on-chip DNA extraction component.

Transcriptional regulation of histidine biosynthesis genes in Corynebacterium glutamicum.

Corynebacterium glutamicum, a gram-positive bacterium, has been widely used for industrial amino acid production. Corynebacterium glutamicum his genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. The latter his operon starts the transcription at the C residue localized 196 bp upstream of the hisD ATG start codon. Our computer-based sequence analysis showed that the region corresponding to the untranslated 5' end of the transcript, named the hisD leader region, displays the typical features of the T-box transcriptional attenuation mechanism. Therefore, expression of the cat reporter gene under the control of the wild-type or mutated hisD leader regions was tested in multi-copy (pProm and pTer series) and in single-copy (pInt series) systems under conditions of sufficient or limited histidine. Our mutational studies led to the conclusion that the CAU histidine specifier and 5'-UGGA-3' sequence in the hisD leader region are required for the hisDCB-orf1-orf2-hisHA-impA-hisFI gene regulation. The cat gene expression from the wild-type leader region was negatively regulated by histidine. However, the cat gene expression from mutated leader regions was irresponsive to the level of histidine in the growth medium. Taken together, we propose that a T-box mediated attenuation mechanism is responsible for the gene expression of the hisDCB-orf1-orf2-hisHA-impA-hisFI operon in C. glutamicum.

Sensitive detection of microRNA using QCM biosensors: sandwich hybridization and signal amplification by TiO2 nanoparticles.

MicroRNA-21 (miR-21) is known to act as an important biomarker for cancer, in that its up-regulation is closely related to several types of malignant tumor. Sensitive and accurate detection of miR-21 using a biosensor is highly challenging. In this study, sensitive and selective detection technology for miR-21 molecules using a quartz crystal microbalance (QCM) biosensor was developed. Sandwich hybridization between miR-21 and specially designed probes and a subsequent TiO2 photocatalytic silver enhancement reaction were the driving forces for sensitive detection with high selectivity for miR-21. Using this strategic approach under optimal conditions, the novel QCM biosensor can detect miR-21 with a LOD of 0.87 pM over the entire linear range from 0.1 pM to 10 µM, with a correlation coefficient of 0.988. In addition, the developed QCM biosensor was very effective in the quantification of miR-21 in serum samples, so the proposed miRNA detection method offers great potential for the diagnosis of early disease, such as cancer and vascular diseases, and could be an excellent alternative for biological research and clinical diagnosis.