A comprehensive Exdia TRF-LFIA for simultaneous quantification of GFAP and NT-proBNP in distinguishing ischemic and hemorrhagic stroke.

The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used. To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

Vertical Magnetic Separation of Circulating Tumor Cells for Somatic Genomic-Alteration Analysis in Lung Cancer Patients.

Efficient isolation and genetic analysis of circulating tumor cells (CTCs) from cancer patients' blood is a critical step for clinical applications using CTCs. Here, we report a novel CTC-isolation method and subsequent genetic analysis. CTCs from the blood were complexed with magnetic beads coated with antibodies against the epithelial cell adhesion molecule (EpCAM) and separated vertically on a density-gradient medium in a modified well-plate. The recovery rate of model CTCs was reasonable and the cell purity was enhanced dramatically when compared to those parameters obtained using a conventional magnetic isolation method. CTCs were recovered from an increased number of patient samples using our magnetic system vs. the FDA-approved CellSearch system (100% vs. 33%, respectively). In 8 of 13 cases, targeted deep sequencing analysis of CTCs revealed private point mutations present in CTCs but not in matched tumor samples and white blood cells (WBCs), which was also validated by droplet digital PCR. Copy-number alterations in CTCs were also observed in the corresponding tumor tissues for some patients. In this report, we showed that CTCs isolated by the EpCAM-based method had complex and diverse genetic features that were similar to those of tumor samples in some, but not all, cases.

Highly dense, optically inactive silica microbeads for the isolation and identification of circulating tumor cells.

Efficient isolation of circulating tumor cells (CTCs) from whole blood is a major challenge for the clinical application of CTCs. Here, we report an efficient method to isolate CTCs from whole blood using highly dense and transparent silica microbeads. The surfaces of silica microbeads were fully covered with an antibody to capture CTCs, and blocked by zwitterionic moieties to prevent the non-specific adsorption of blood cells. Owing to the high density of the silica microbeads, the complexation of CTCs with silica microbeads resulted in the efficient sedimentation of CTC-microbead complexes, which enabled their discrimination from other blood cells in density gradient media. Model CTCs (MCF-7, HCC827, and SHP-77) with various levels of epithelial cell adhesion molecule (EpCAM) were isolated efficiently, especially those with low EpCAM expression (SHP-77). Moreover, the transparency of silica microbeads enabled CTCs to be clearly identified without interference caused by microbeads. The improved sensitivity resulted in increased CTC recovery from patient samples compared with the FDA-approved CellSearch system (14/15 using our method; 5/15 using the CellSearch system). These results indicate that the isolation method described in this report constitutes a powerful tool for the isolation of CTCs from whole blood, which has important applications in clinical practice.

Luminol chemiluminescence biosensor for glycated hemoglobin (HbA1c) in human blood samples.

Luminol chemiluminescence (CL) biosensor based on boronic acid modified gold substrate has been developed for the determination of glycated hemoglobin (HbA1c) in human blood samples. In order to selectively capture HbA1c in sample, carboxy-EG6-undecanethiol was self-assembled on a gold thin-film substrate, followed by covalent coupling of 3-aminophenyl boronic acid (3-APBA). The captured HbA1c containing four iron heme groups plays as a catalyst for luminol CL reaction in the presence of hydrogen peroxide, and thus the luminol CL response is linearly proportional to the amount of HbA1c captured on the biosensor surface. The present biosensor showed linear dynamic range of HbA1c from 2.5% to 17.0%, which well covers the clinically important concentration range. In addition, the present biosensor exhibited negligible response to interfering species such as hemoglobin, fructose, and sorbitol. The present HbA1c biosensor was applied to the determination of HbA1c in human blood samples and the results were well agreed with that obtained with a conventional method.

A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads.

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.

Fully automated circulating tumor cell isolation platform with large-volume capacity based on lab-on-a-disc.

Full automation with high purity for circulating tumor cell (CTC) isolation has been regarded as a key goal to make CTC analysis a "bench-to-bedside" technology. Here, we have developed a novel centrifugal microfluidic platform that can isolate the rare cells from a large volume of whole blood. To isolate CTCs from whole blood, we introduce a disc device having the biggest sample capacity as well as manipulating blood cells for the first time. The fully automated disc platform could handle 5 mL of blood by designing the blood chamber having a triangular obstacle structure (TOS) with lateral direction. To guarantee high purity that enables molecular analysis with the rare cells, CTCs were bound to the microbeads covered with anti-EpCAM to discriminate density between CTCs and blood cells and the CTCs being heavier than blood cells were only settled under a density gradient medium (DGM) layer. To understand the movement of CTCs under centrifugal force, we performed computational fluid dynamics simulation and found that their major trajectories were the boundary walls of the DGM chamber, thereby optimizing the chamber design. After whole blood was inserted into the blood chamber of the disc platform, size- and density-amplified cancer cells were isolated within 78 min, with minimal contamination as much as approximately 12 leukocytes per milliliter. As a model of molecular analysis toward personalized cancer treatment, we performed epidermal growth factor receptor (EGFR) mutation analysis with HCC827 lung cancer cells and the isolated cells were then successfully detected for the mutation by PCR clamping and direct sequencing.

Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles.

Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs.

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.

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.

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter.

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 µm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.

Centrifugal microfluidics for biomedical applications.

The centrifugal microfluidic platform has been a focus of academic and industrial research efforts for almost 40 years. Primarily targeting biomedical applications, a range of assays have been adapted on the system; however, the platform has found limited commercial success as a research or clinical tool. Nonetheless, new developments in centrifugal microfluidic technologies have the potential to establish wide-spread utilization of the platform. This paper presents an in-depth review of the centrifugal microfluidic platform, while highlighting recent progress in the field and outlining the potential for future applications. An overview of centrifugal microfluidic technologies is presented, including descriptions of advantages of the platform as a microfluidic handling system and the principles behind centrifugal fluidic manipulation. The paper also discusses a history of significant centrifugal microfluidic platform developments with an explanation of the evolution of the platform as it pertains to academia and industry. Lastly, we review the few centrifugal microfluidic-based sample-to-answer analysis systems shown to date and examine the challenges to be tackled before the centrifugal platform can be more broadly accepted as a new diagnostic platform. In particular, fully integrated, easy to operate, inexpensive and accurate microfluidic tools in the area of in vitro nucleic acid diagnostics are discussed.

A fully automated immunoassay from whole blood on a disc.

A portable, disc-based, and fully automated enzyme-linked immuno-sorbent assay (ELISA) system is developed to test infectious diseases from whole blood. The innovative laser irradiated ferrowax microvalves and centrifugal microfluidics were utilized for the full integration of microbead-based suspension ELISA assays on a disc starting from whole blood. The concentrations of the antigen and the antibody of Hepatitis B virus (HBV), HBsAg and Anti-HBs respectively, were measured using the lab-on-a-disc (LOD). All the necessary reagents are preloaded on the disc and the total process of the plasma separation, incubation with target specific antigen or antibody coated microbeads, multiple steps of washing, enzyme reaction with substrates, and the absorbance detection could be finished within 30 minutes. Compared to the conventional ELISA, the operation time was dramatically reduced from over 2 hours to less than 30 minutes while the limit of detection was kept similar; e.g. the limit of detection of Anti-HBs tests were 8.6 mIU mL(-1) and 10 mIU mL(-1) for the disc-based and the conventional ELISA respectively.

Gold nanoparticles for one step DNA extraction and real-time PCR of pathogens in a single chamber.

The optothermal properties of nanoparticles are of interest for biosensors and highly sensitive biochip applications. In this respect, the longitudinal resonance of Au nanorods was used to transform near infrared energy into thermal energy in a microfluidic chip. The resulting heat generated effectively caused pathogen lysis. Consequently the DNA was extracted out of the cell body and transferred to a PCR system. This resulted in the successful demonstration of a one step real-time PCR system for pathogen detection without removal or changing of reagents.

Multifunctional microvalves control by optical illumination on nanoheaters and its application in centrifugal microfluidic devices.

Valving is critical in microfluidic systems. Among many innovative microvalves used in lab-on-a-chip applications, phase change based microvalves using paraffin wax are particularly attractive for disposable biochip applications because they are simple to implement, cost-effective and biocompatible. However, previously reported paraffin-based valves require embedded microheaters and therefore multi-step operation of many microvalves was a difficult problem. Besides, the operation time was relatively long, 2-10 s. In this paper, we report a unique phase change based microvalve for rapid and versatile operation of multiple microvalves using a single laser diode. The valve is made of nanocomposite materials in which 10 nm-sized iron oxide nanoparticles are dispersed in paraffin wax and used as nanoheaters when excited by laser irradiation. Laser light of relatively weak intensity was able to melt the paraffin wax with the embedded iron oxide nanoparticles, whereas even a very intense laser beam does not melt wax alone. The microvalves are leak-free up to 403.0 +/- 7.6 kPa and the response times to operate both normally closed and normally opened microvalves are less than 0.5 s. Furthermore, a sequential operation of multiple microvalves on a centrifugal microfluidic device using a single laser diode was demonstrated. It showed that the optical control of multiple microvalves is fast, robust, simple to operate, and requires minimal chip space and thus is well suited for fully integrated lab-on-a-chip applications.

One-step pathogen specific DNA extraction from whole blood on a centrifugal microfluidic device.

We report a fully integrated, pathogen-specific DNA extraction device utilizing centrifugal microfluidics on a polymer based CD platform. By use of the innovative laser irradiated Ferrowax microvalve (LIFM) together with the rapid cell lysis method using laser irradiation on magnetic particles, we could, for the first time, demonstrate a fully integrated pathogen specific DNA extraction from whole blood on a CD. As a model study, DNA extraction experiments from whole blood spiked with Hepatitis B virus (HBV) and E.coli were conducted. The total process of the plasma separation, mixing with magnetic beads conjugated with target specific antibodies, removal of plasma residual, washing and DNA extraction was finished within 12 min with only one manual step, the loading of 100 microL of whole blood. Real-time PCR results showed that the concentration of DNA prepared on a CD using a portable sample preparation device was as good as those by conventional bench top protocol. It demonstrates that our novel centrifugal microfluidics platform enables a full integration of complex biological reactions that require multi-step fluidic control.