Microchip Cytometry for Multiplexed Single-Cell Protein Detection in a Low-Resource Setting toward Point of Care Diagnosis.
ABSTRACT: Multiplex measurement of protein expression with the single-cell resolution has been challenging. Although a few conventional approaches including flow cytometry and immunofluorescence-based methods have been developed to detect proteins in individual cells, they are either dependent on bulky instrument or not multiplexed and high-throughput enough. Here we present a portable single-cell analysis system that is operable in a resource-limited environment. A stand-sit microchip housed in a clamp enables simple and instrument-free operation of all necessary steps, and the detection based on immunogold enhancement exonerates the reliance on fluorescence optics and electronics. The quantified sensitivity was found comparable to the conventional fluorescence approaches. We used this system to analyze five immune effector proteins and found the system is equally effective to detect those proteins in hundreds of single cells. Significant increase of cytokine protein production by THP1 monocytes was observed upon stimulation by lipopolysaccharide. Further study showed that a low-end imaging setup with low resolution can also detect signals without much loss of sensitivity. Taken together, this portable multiplex single-cell system may find broad biomedical applications in a field setting.
Project description:Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3) in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.
Project description:The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in<i>-vitro</i> crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.
Project description:Ovarian cancer is asymptomatic in the early stages and most patients present with advanced levels of disease. The lack of cost-effective methods that can achieve frequent, simple and non-invasive testing hinders early detection and causes high mortality in ovarian cancer patients. Here, we report a simple and inexpensive microchip ELISA-based detection module that employs a portable detection system, i.e., a cell phone/charge-coupled device (CCD) to quantify an ovarian cancer biomarker, HE4, in urine. Integration of a mobile application with a cell phone enabled immediate processing of microchip ELISA results, which eliminated the need for a bulky, expensive spectrophotometer. The HE4 level detected by a cell phone or a lensless CCD system was significantly elevated in urine samples from cancer patients (n = 19) than healthy controls (n = 20) (p < 0.001). Receiver operating characteristic (ROC) analyses showed that the microchip ELISA coupled with a cell phone running an automated analysis mobile application had a sensitivity of 89.5% at a specificity of 90%. Under the same specificity, the microchip ELISA coupled with a CCD had a sensitivity of 84.2%. In conclusion, integration of microchip ELISA with cell phone/CCD-based colorimetric measurement technology can be used to detect HE4 biomarker at the point-of-care (POC), paving the way to create bedside technologies for diagnostics and treatment monitoring.
Project description:A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used).
Project description:Cellular heterogeneity in function and response to therapeutics has been a major challenge in cancer treatment. The complex nature of tumor systems calls for the development of advanced multiplexed single-cell tools that can address the heterogeneity issue. However, to date such tools are only available in a laboratory setting and don't have the portability to meet the needs in point-of-care cancer diagnostics. Towards that application, we have developed a portable single-cell system that is comprised of a microchip and an adjustable clamp, so on-chip operation only needs pipetting and adjusting of clamping force. Up to 10 proteins can be quantitated from each cell with hundreds of single-cell assays performed in parallel from one chip operation. We validated the technology and analyzed the oncogenic signatures of cancer stem cells by quantitating both aldehyde dehydrogenase (ALDH) activities and 5 signaling proteins in single MDA-MB-231 breast cancer cells. The technology has also been used to investigate the PI3K pathway activities of brain cancer cells expressing mutant epidermal growth factor receptor (EGFR) after drug intervention targeting EGFR signaling. Our portable single-cell system will potentially have broad application in the preclinical and clinical settings for cancer diagnosis in the future.
Project description:<h4>Background</h4>CD4(+) T-lymphocyte count (CD4 count) is a standard method used to monitor HIV-infected patients during anti-retroviral therapy (ART). The World Health Organization (WHO) has pointed out or recommended that a handheld, point-of-care, reliable, and affordable CD4 count platform is urgently needed in resource-scarce settings.<h4>Methods</h4>HIV-infected patient blood samples were tested at the point-of-care using a portable and label-free microchip CD4 count platform that we have developed. A total of 130 HIV-infected patient samples were collected that included 16 de-identified left over blood samples from Brigham and Women's Hospital (BWH), and 114 left over samples from Muhimbili University of Health and Allied Sciences (MUHAS) enrolled in the HIV and AIDS care and treatment centers in the City of Dar es Salaam, Tanzania. The two data groups from BWH and MUHAS were analyzed and compared to the commonly accepted CD4 count reference method (FACSCalibur system).<h4>Results</h4>The portable, battery operated and microscope-free microchip platform developed in our laboratory (BWH) showed significant correlation in CD4 counts compared with FACSCalibur system both at BWH (r?=?0.94, p<0.01) and MUHAS (r?=?0.49, p<0.01), which was supported by the Bland-Altman methods comparison analysis. The device rapidly produced CD4 count within 10 minutes using an in-house developed automated cell counting program.<h4>Conclusions</h4>We obtained CD4 counts of HIV-infected patients using a portable platform which is an inexpensive (<$1 material cost) and disposable microchip that uses whole blood sample (<10 µl) without any pre-processing. The system operates without the need for antibody-based fluorescent labeling and expensive fluorescent illumination and microscope setup. This portable CD4 count platform displays agreement with the FACSCalibur results and has the potential to expand access to HIV and AIDS monitoring using fingerprick volume of whole blood and helping people who suffer from HIV and AIDS in resource-limited settings.
Project description:Extracellular vesicles (EVs) are promising novel cancer biomarkers. However, rapid and easy analysis of EVs is challenging because conventional detection methods require large sample volumes and long detection times. Microchip-based analytical systems have particularly attracted attention for development of point-of-care (POC) diagnostics. Previously, various biomarker detection methods on a portable power-free poly(dimethylsiloxane) (PDMS) microchip using laminar flow-assisted dendritic amplification have been developed. Recently, for easy functionalization, we proposed a microchannel inner surface-functionalized power-free PDMS microchip (SF-PF microchip) utilizing electron beam-induced graft polymerization. In this study, we apply the technique and prepare a novel SF-PF microchip. On the microchip, EVs were successfully detected. The required sample volume was 1.0 ?L, and the total analysis time was 20 min. The microchip can contribute to EV-based POC cancer diagnosis.
Project description:Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine) with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231) are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclin-dependent kinase inhibitor 1A (CDKN1A), and aurora kinase A (AURKA) genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation.
Project description:A plug-in electrophoresis microchip for large-scale use aimed at improving maintainability with low fabrication and maintenance costs is proposed in this paper. The plug-in microchip improves the maintainability of a device because the damaged microchannel layer can be changed without needing to cut off the circuit wires in the detection component. Obviously, the plug-in structure reduces waste compared with earlier microchips; at present the whole microchip has to be discarded, including the electrode layer and the microchannel layer. The fabrication cost was reduced as far as possible by adopting a steel template and printed circuit board electrodes that avoided the complex photolithography, metal deposition and sputtering processes. The detection performance of our microchip was assessed by electrophoresis experiments. The results showed an acceptable gradient and stable detection performance. The effect of the installation shift between the microchannel layer and the electrode layer brought about by the plug-in structure was also evaluated. The results indicated that, as long as the shift was controlled within a reasonable scope, its effect on the detection performance was acceptable. The plug-in microchip described in this paper represents a new train of thought for the large-scale use and design of portable instruments with electrophoresis microchips in the future.
Project description:Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.