Microfluidic platforms for discovery and detection of molecular biomarkers

Microfluidics has emerged as a promising platform for discovery and detection of molecular biomarkers recently. With this approach, the discovery of these biomarkers could be more efficient in time and consumes less samples and reagents. Furthermore, the entire discovery process could be automated since all the functional microfluidic devices such as micropumps and microvalves could be integrated on a single chip. Similarly, the detection of the discovered molecular biomarkers is also promising. Detection of nucleic acid biomarkers, protein biomarkers, and metabolite biomarkers has been demonstrated on microfluidic platforms recently. When compared with their large-scale counterparts, the miniature system can perform the detection of these biomarkers within less analysis time while a multiplexed detection scheme could be easily achieved. Furthermore, the entire detection process could be automated on the single chip as well. This review paper is therefore to review the recent development of microfluidic devices and systems for the discovery and detection of the molecular biomarker. Techniques for biomarker discovery, verification, and detection that have been adapted into microfluidics were first reviewed, and their advantages were highlighted. The new approach of biomarker screening based on in vitro-generated affinity reagents such as nucleic acid aptamers and peptide affinity reagents was then reviewed. Finally, in the biomarker detection section, this review placed a special emphasis on commercialized microfluidic-based diagnostics for molecular biomarkers.     

An automatic microfluidic system for rapid screening of cancer stem-like cell-specific aptamers

This study reports a microfluidic system which automatically performs the systematic evolution of ligands by exponential enrichment (SELEX) process for rapid screening of aptamers which are specific to cancer stem-like cells. The system utilizes magnetic bead-based techniques to select DNA aptamers and has several advantages including a rapid, automated screening process, and less consumption of cells and reagents. By integrating a microfluidic control module, a magnetic bead-based aptamer extraction module, and a temperature control module, the entire Cell-SELEX process can be performed in a shorter period of time. Compared with the traditional Cell-SELEX process, this microfluidic system is more efficient and consumes fewer sample volumes. It only takes approximately 3 days for an entire Cell-SELEX process with 15 screening runs, which is relatively faster than that of a traditional Cell-SELEX process (1 week for 15 rounds). The binding affinity of this resulting specific aptamer was measured by a flow cytometric analysis to have a dissociation constant (K _d) of 15.32 nM. The capture rate for cancer stem-like cells using the specific aptamer-conjugated bead is better than that using Ber-EP4 antibody-conjugated bead. This microfluidic system may provide a powerful platform for the rapid screening of cell-specific aptamers.     

An automatic microfluidic system that continuously performs the systematic evolution of ligands by exponential enrichment

The systematic evolution of ligands by exponential enrichment (SELEX) technique has been extensively used to screen molecule-specific aptamers from combinatorial libraries of synthetic nucleic acids. Aptamers are single-stranded DNA or RNA, which have a high affinity to a large variety of molecules ranging from small drugs or metabolites to cells. Therefore, they have a variety of promising applications such as for diagnostics and targeted therapeutics. In this study, a new microfluidic chip was developed to perform continuous screening of DNA-based aptamers in an automatic format. When compared with the existing manual procedure, the developed microfluidic chip has several advantages including a rapid and efficient screening process, automation, and less consumption of samples/reagents. Experimental data showed that an aptamer specific to alpha-fetoprotein was successfully screened from a random DNA pool. The entire screening process (five continuous, repetitive rounds) can be completed within 6?h, which is much faster than the traditional methods (more than 15?h). An automatic, rapid and efficient SELEX process was performed by this developed microfluidic chip, which may enable a generalized platform for the fast screening of DNA-based biomarkers in the future.     

应用于肿瘤细胞基因表达水平分析的微流控芯片

近年来快速发展的微流控技术为单细胞基因诊断的基础研究提供了一个有力的平台,微流控芯片具有样品和试剂低消耗、快速实时、大量样本平行处理等优点,还可以使分析过程自动化、防止污染以及完成自动高效的重复实验。本文主要介绍一种用于高通量单细胞基因表达水平分析的微流控芯片技术平台。芯片集成了单细胞捕获、裂解、纯化、逆转录等生物样品处理单元,通过内置气动微阀实现细胞反应腔的独立寻址,从而实现单细胞处理与分析整个过程的自动化操控。实验结果显示了此平台可以实现基因表达水平稳定性的评估,具有应用于肿瘤早期诊断领域的潜力。     

The synergistic effect of periodic immunomagnetics and microfluidics on universally capturing circulating tumor cells

Efficient capture data on circulating tumor cells (CTCs) determines early-stage cancer diagnosis and contributes to timely clinical treatment. We present an enhanced method of capturing CTCs accomplished by a microfluidic device integrated with magnetic field to activate the kinetic motion of in-device magnetic beads. The device, consisting of a microfluidic chamber and two electrode chips applied with pulsatile alternating current in both, is designed based on simulations on the periodicity characteristic of magnetic beads as well as the effect of heat dissipation on cell culture medium manifest. Using MEMS technologies, the prototype is fabricated and assembled. The cell capture experiments based on active magnetic beads are achieved in separation of rare cancer cells (MCF7 cells) with low concentration. The capture rate is estimated up to 88 %, with great potential of dramatically improving detection efficiency in disease diagnostics.     

A novel integrated microfluidic platform to perform fluorescence in situ hybridization for chromosomal analysis

The fluorescence in situ hybridization (FISH) technique has been commonly employed to detect the chromosomal abnormalities. However, applications of this technique are limited due to its lengthy process and labor-intensive sample preparation. In this study, a novel integrated microfluidic chip capable of performing the entire FISH protocol automatically was reported. This novel technique can achieve several advantages, including reduce the consumption of bio-samples and reagents, automation and rapid analysis compared to the conventional method. In this study, several functional microfluidic devices were integrated on a single chip to perform automatic FISH on the microfluidic platform. Experimental data demonstrated that the developed microfluidic system successfully provided superior performance for probing the chromosomal abnormality of cells. Furthermore, the novel microfluidic system performed the entire process automatically within 3 h, where the conventional method required 10 h to perform the entire protocol manually. This data indicated superior performance of the novel method. Our findings conclude that the novel integrated FISH protocol is more convenient to perform large quantities of samples, which can be used in clinical trials.     

Discovery of diagnostic serum biomarkers of gastric cancer using proteomics

Gastric cancer has significant morbidity and mortality worldwide and locally. Good prognosis relies on an early diagnosis. However, this remains a challenge due to the lack of specific and sensitive serum biomarkers for early detection. Hence, there is a constant search for these biomarkers for screening purposes. Proteomic profiling enables a new approach to the discovery of biomarkers in disease. This review presents recent attempts in search of gastric cancer serum biomarker using proteomics. Different methodologies and different types of samples were employed by different groups of researchers. Major difficulties were encountered in the discovery processes, including interference from abundant proteins and continuous changing serum proteomes from different individuals.     

A DNA methylation assay for detection of ovarian cancer cells using a HpaII/MspI digestion-based PCR assay in an integrated microfluidic system

Early and accurate diagnosis of cancer plays a very important role in favorable clinical outcomes. DNA methylation of tumor suppressor genes has been recognized as a diagnostic biomarker for early carcinogenesis. The presence of 5-methylcytosine in the CpG islands in the promoter region of a tumor suppressor gene is an important indicator of DNA methylation. However, the standard detection assay utilizing a bisulfite treatment and HpaII/MspI endonuclease digestion is a tedious and lengthy process and requires a relatively large amount of DNA for testing. In this study, the methylated DNAs of various tumor suppressor genes, HAAO, HOXA9 and SFRP5, were chosen as candidates for detection of ovarian cancer cells. The entire experimental process for the DNA methylation assay, including target DNA isolation, HpaII/MspI endonuclease digestion, and nucleic acid amplification has been realized in an integrated microfluidic system. The limit of detection using this developed system has been experimentally determined to be 10² cells/reaction. The entire process from sample loading to analysis of the results only took 3 h which is much faster than the existing protocols. Different sources of biosamples, such as cells, ascites and serums, could be detected with the methylated DNA, indicating that this developed microfluidic system could be adapted for clinical use. Thus, this developed microsystem may be a promising platform for the rapid and early diagnosis of cancers.     

Observation of peptide differences between cancer and control in gastric juice

Biomarkers for various diseases have been extensively searched for the past 5 years. Nevertheless, most efforts were focused on the search for protein biomarkers from serum samples. In this work, we tried to look for peptide biomarkers from gastric juice samples with MALDI‐TOF‐MS. More than 200 gastric juice samples from healthy people, gastric ulcer patients, duodenal ulcer patients, and cancer patients were examined. There were clear pattern differences of mass spectra among samples from healthy people and patients with different gastric diseases. We found five peptides for gastric cancer diagnosis with high sensitivity and specificity. Sequences of these five peptides, including two pepsinogen fragments, leucine zipper protein fragment, albumin fragment, and α‐1‐antitrypsin fragment, have been identified by mass spectrometric analysis and immuno‐deplete assay with antibodies.     

Quantitative proteomics for identification of cancer biomarkers

Quantitative proteomics can be used for the identification of cancer biomarkers that could be used for early detection, serve as therapeutic targets, or monitor response to treatment. Several quantitative proteomics tools are currently available to study differential expression of proteins in samples ranging from cancer cell lines to tissues to body fluids. 2-DE, which was classically used for proteomic profiling, has been coupled to fluorescence labeling for differential proteomics. Isotope labeling methods such as stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tagging (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ), and (18) O labeling have all been used in quantitative approaches for identification of cancer biomarkers. In addition, heavy isotope labeled peptides can be used to obtain absolute quantitative data. Most recently, label-free methods for quantitative proteomics, which have the potential of replacing isotope-labeling strategies, are becoming popular. Other emerging technologies such as protein microarrays have the potential for providing additional opportunities for biomarker identification. This review highlights commonly used methods for quantitative proteomic analysis and their advantages and limitations for cancer biomarker analysis.     

Microfluidic systems for extracting nucleic acids for DNA and RNA analysis

This paper is to review the differences in the developments of microfluidic chips for extracting genomic deoxyribonucleic acid (DNA) and viral ribonucleic acid (RNA) from blood by the Biosensor Focus Interest Group (BFIG) in Singapore. DNA was extracted in a multi-step process by isolating and lysing white blood cells (WBC), typically ∼10 μm in diameter. Viral RNA was extracted directly from the submicron viruses in the blood. In terms of basic microfluidic components required, both DNA and RNA extractions used similar mixers for mixing reagents, filters for capturing or separating the blood cells, and a binder for capturing and purifying the DNA/RNA molecules. The designs of the filters were adapted to either capture WBC for DNA isolation or capture all virus particles for RNA isolation. The designs of these two kinds of filters had to be different. Besides the differences in the sizes of WBC and viruses, the concentration of the virus particles is usually much lower than WBC. Thus, a much higher volume of blood for filtering would be required for extracting viral RNA, especially for the intention to detect the viruses at early onset of infection. With proper modifications of the protocols, it has been demonstrated that both genomics DNA and viral RNA could be extracted successfully in these microfluidic chips. The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions.     

Biomarkers of colorectal cancer: Recent advances and future challenges

Colorectal cancer (CRC) is a common malignancy and it contributes significantly to cancer mortality. Outcomes in colorectal cancer vary between patients and this is due to the complexity of colorectal carcinogenesis. Interactions between tumor cells and their microenvironment, genetic alterations, and changes in intracellular signalling networks are just some of the abnormal pathways involved in colorectal cancer development. Recent research has targeted components of all of these systems in order to develop biomarkers to aid in the early diagnosis of CRC and to assist in prognostic stratification. Proteomic analysis of tissue or blood-derived samples from CRC patients has proven to be a valuable technique for the identification of potentially informative biomarkers. Such biomarkers may prove to be clinically applicable and could offer greater patient acceptability when compared to conventional methods such as fecal-based testing. In this article we review the recent advances in the development of protein biomarkers of CRC with an emphasis on biomarkers available in the patient's serum and from tissue-based samples. Future challenges in terms of the development of accurate diagnostic, prognostic, and predictive biomarkers of CRC and the importance of validation and patient acceptability are also discussed.     

An integrated microfluidic system for live bacteria detection from human joint fluid samples by using ethidium monoazide and loop-mediated isothermal amplification

Periprosthetic joint infection (PJI) is one of the severe complications of prosthetic joint replacement. Delayed PJI diagnosis may anchor bacteria in periprosthetic tissues, and removal of the prosthesis might be inevitable. The diagnosis of PJI depends on the identification of microorganisms by standard microbiological cultures or more advanced molecular diagnostic methods for detection of bacterial genes. However, these methods are relatively time-consuming, labor-intensive and not human error-free. Moreover, it is challenging to distinguish live from dead bacteria by using DNA-based molecular diagnostics since bacterial DNA will be remained in the tissue even after the death of the bacteria. In this work, an integrated microfluidic system has been developed to perform the entire molecular diagnostic process for the PJI diagnosis in a single chip. We combined the loop-mediated isothermal amplification (LAMP) with ethidium monoazide (EMA) in an integrated microfluidic system to identify live bacteria with reasonable sensitivity and high specificity. All the diagnostic processes including bacteria isolation, cell lysis, DNA amplification and optical detection can be automatically performed on the integrated microfluidic system by using a compact custom-made control system. The integrated system can accommodate four primers complementary to six regions of the target genes and improve the detection limit by using LAMP. The limit of detection in this multiple EMA-LAMP assay could be as low as 5 fg/reaction (~1 CFU/reaction) when choosing an optimized primer set as we demonstrated in mecA gene detection. Thus, the developed system for PJI diagnosis has great potential to become a point-of-care device.     

An integrated microfluidic system for the determination of microalbuminuria by measuring the albumin-to-creatinine ratio

This study presents an integrated microfluidic system for the determination of microalbuminuria (MAU) through the measurements of the albumin-to-creatinine ratios in patients’ urinary samples. Albumin concentrations are determined based on a non-immunological dye binding assay in which the dyes react specifically with albumin to undergo a strong fluorescence enhancement. Creatinine concentrations are determined based on the Jaffé reaction in which the reagents react specifically with creatinine to form orange–red colored complexes. Two calibration curves for determining the concentrations of urinary albumin and creatinine are constructed with assay ranges of 5–220 and 1–100 mg/l, respectively. Using this system to determine the ACRs of collected clinical urine samples, statistical tools including Bland–Altman bias plot and Passing–Bablok regression analysis show that the results obtained by the proposed microfluidic system are in good agreement with those obtained by conventional methods. This simple, automatic, inexpensive, and microchip-based platform demonstrates a promising alternative to the conventional assays for determining MAU and may be suited for clinical applications.     

Proteomic and other analyses to determine the functional consequences of deregulated kallikrein-related peptidase (KLK) expression in prostate and ovarian cancer

Rapidly developing proteomic tools are improving detection of deregulated kallikrein-related peptidase (KLK) expression, at the protein level, in prostate and ovarian cancer, as well as facilitating the determination of functional consequences downstream. MS-driven proteomics uniquely allows for the detection, identification, and quantification of thousands of proteins in a complex protein pool, and this has served to identify certain KLKs as biomarkers for these diseases. In this review, we describe applications of this technology in KLK biomarker discovery and elucidate MS-based techniques that have been used for unbiased, global screening of KLK substrates within complex protein pools. Although MS-based KLK degradomic studies are limited to date, they helped to discover an array of novel KLK substrates. Substrates identified by MS-based degradomics are reported with improved confidence over those determined by incubating a purified or recombinant substrate and protease of interest, in vitro. We propose that these novel proteomic approaches represent the way forward for KLK research, in order to correlate proteolysis of biological substrates with tissue-related consequences, toward clinical targeting of KLK expression and function for cancer diagnosis, prognosis, and therapies.     

Fabrication techniques to realize CMOS-compatible microfluidicmicrochannels

With microfluidic systems becoming more prominent, fabrication techniques for microfluidic systems are increasingly more important. An interesting alternative to existing fabrication techniques is to embed fluidic systems within an integrated circuit by micromachining materials in the integrated circuit itself. This paper describes novel methods for fabricating one component in the complementary metal-oxide-semiconductor (CMOS) microfluidic system, the microchannel. These techniques allow direct integration of sensors, actuators, or other electronics with the microchannel. This method expands the functional applications for microfluidic systems beyond their current abilities. By utilizing the methods described within this paper, a complete “smart” microfluidic system could be batch fabricated on a single integrated circuit (IC) chip