Ultrasensitive detection of small biomolecules using aptamer-based molecular recognition and nanoparticle counting.

Detection of small biomolecules is critical for understanding molecular mechanisms in biological systems and performing in vitro diagnosis in clinics. Current antibody based detection methods face large challenges in detecting small biomolecules at low concentrations. We report a new method for detecting small biomolecules based on molecular recognition and nanoparticle (NP) counting. Aptamer-functionalized NPs are attached to complementary sequence (CS)-conjugated microparticle (MP) carriers. In the presence of target small biomolecules at ultra low concentrations, NPs would be released from the MP carriers. Coupled with a resistive pulse sensor (RPS) using a micropore that counts the released NPs, this method can measure the concentrations of target biomolecules at low concentrations with high sensitivity and high throughput. Adenosine was used as a model to demonstrate the feasibility of this method. It is demonstrated that this method can detect a wide range of adenosine concentrations with a low detection limit of 0.168 nM, which is 10 times lower than that of the ELISA kit. With its simple structure, high sensitivity, and high reproducibility, this detection method holds great potential for the ultrasensitive detection of low abundance small biomolecules.

Efficient microfluidic enrichment of nano-/submicroparticle in viscoelastic fluid.

The enrichment and focusing of the nano-/submicroparticle (e.g., 150-1000 nm microvesicle shed from the plasma membrane) in the viscoelastic fluid has great potentials in the biomedical and clinical applications such as the disease diagnosis and the prognostic test for liquid biopsy. However, due to the small size and the resulting weak hydrodynamic force, the efficient manipulation of the nano-/submicroparticle by the passive viscoelastic microfluidic technology remains a major challenge. For instance, a typically long channel length is often required to achieve the focusing or the separation of the nano-/submicroparticle, which makes it difficult to be integrated in small chip area. In this work, a microchannel with gradually contracted cross-section and high aspect ratio (the ratio of the height to the average width of channel) is utilized to enhance the hydrodynamic force and change the force direction, eventually leading to the efficient enrichment of nano-/submicroparticles (500 and 860 nm) in a short channel length (2 cm). The influence of the flow rate, the particle size, the solid concentration, and the channel geometry on the enrichment of the nano-/submicroparticles are investigated. With simple structure, small footprint, easy operation, and good performance, the present device would be a promising platform for various lab-chip microvesicle-related biomedical research and disease diagnosis.

A Microfluidic Sensor for Continuous, in Situ Surface Charge Measurement of Single Cells.

Cell surface charge has been recognized as an important cellular property. We developed a microfluidic sensor based on resistive pulse sensing to assess surface charge and sizes of single cells suspended in a continuous flow. The device consists of two consecutive resistive pulse sensors (RPSs) with identical dimensions. Opposite electric fields were applied on the two RPSs. A charged cell in the RPSs was accelerated or decelerated by the electric fields and thus exhibited different transit times passing through the two RPSs. The cell surface charge is measured with zeta potential that can be quantified with the transit time difference. The transit time of each cell can be accurately detected with the width of pulses generated by the RPS, while the cell size can be calculated with the pulse magnitude at the same time. This device has the ability to detect surface charges and sizes of individual cells with high tolerance in cell types and testing solutions compared with traditional electrophoretic light scattering methods. Three different types of cells including HeLa cancer cells, human dermal fibroblast cells, and human umbilical vein endothelial cells (HUVECs) were tested with the sensor. Results showed a significant difference of zeta potentials between HeLa cells and fibroblasts or HUVECs. In addition, when HeLa cells were treated with various concentrations of glutamine, the effects on cancer cell surface charge were detected. Our results demonstrated the great potential of using our sensor for cell type sorting, cancer cell detection, and cell status analysis.

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection.

We report a high-sensitivity cell secretome detection method using competitive immuno-aggregation and a micro-Coulter counter. A target cell secretome protein competes with anti-biotin-coated microparticles (MPs) to bind with a biotinylated antibody (Ab), causing decreased aggregation of the functionalized MPs and formation of a mixture of MPs and aggregates. In comparison, without the target cell secretome protein, more microparticles are functionalized, and more aggregates are formed. Thus, a decrease in the average volume of functionalized microparticles/aggregates indicates an increase in cell secretome concentration. This volume change is measured by the micro-Coulter counter, which is used to quantitatively estimate the cell secretome concentration. Vascular endothelial growth factor (VEGF), one of the key cell secretome proteins that regulate angiogenesis and vascular permeabilization, was used as the target protein to demonstrate the sensing principle. A standard calibration curve was generated by testing samples with various VEGF concentrations. A detection range from 0.01 ng/mL to 100.00 ng/mL was achieved. We further demonstrated the quantification of VEGF concentration in exogenous samples collected from the secretome of human mesenchymal stem cells (hMSCs) at different incubation times. The results from the assay agree well with the results of a parallel enzyme-linked immunoabsorbent assay (ELISA) test, indicating the specificity and reliability of the competitive immuno-aggregation assay. With its simple structure and easy sample preparation, this assay not only enables high sensitivity detection of VEGF but also can be readily extended to other types of cell secretome analysis as long as the specific Ab is known.

Development and Comparison of Two Immuno-disaggregation Based Bioassays for Cell Secretome Analysis.

Cell secretome analysis has gained increasing attention towards the development of effective strategies for disease treatment. Analysis of cell secretome enables the platform to monitor the status of disease progression, facilitating therapeutic outcomes. However, cell secretome analysis is very challenging due to its versatile and dynamic composition. Here, we report the development of two immuno-disaggregation bioassays using functionalized microparticles for the quantitative analysis of the cell secretome. Methods: We evaluated the feasibility of our developed immuno-disaggregation bioassays using antibody-conjugated MPs and protein-conjugated MPs for the detection of target cell secretome protein. The vascular endothelial growth factor (VEGF)-165 protein was tested as a model cell secretome protein in the serum and serum-free conditions. The status of MP aggregates was examined with a light microscopy and AccuSizerTM 780 Optical Particle Sizer. The accuracy of our bioassays measurement was compared with standard ELISA method. Results: The developed bioassays successfully detected target VEGF protein present in serum-free buffer and serum-containing complete cell culture medium with high sensitivity and specificity. Additionally, the immuno-disaggregation bioassays using antibody-conjugated MPs and protein-conjugated MPs have a wide detection range from 0.01 ng/mL to 100 ng/mL and 0.5 ng/mL to 100 ng/mL, respectively. The sensitivity of the bioassay using antibody-conjugated MPs was approximately one order of magnitude higher than the bioassay using protein-conjugated MPs. Conclusion: Our promising results indicate the potential of the developed bioassays as powerful platforms for the quantitative analysis of cell secretome.

Structure-Function Relationships of a Tertiary Amine-Based Polycarboxybetaine.

Zwitterionic polycarboxybetaine (PCB) materials have attracted noticeable interest for biomedical applications, such as wound healing/tissue engineering, medical implants, and biosensors, due to their excellent antifouling properties and design flexibility. Antifouling materials with buffering capability are particularly useful for many biomedical applications. In this work, an integrated zwitterionic polymeric material, poly(2-((2-hydroxyethyl)(2-methacrylamidoethyl)ammonio)acetate) (PCBMAA-1T), was synthesized to carry desired properties (antifouling, switchability and buffering capability). A tertiary amine was used to replace quaternary ammonium as the cation to endow the materials with buffering capability under neutral pH. Through this study, a better understanding on the structure-property relationship of zwitterionic materials was obtained. The tertiary amine cation does not compromise antifouling properties of zwitterionic materials. The amount of adsorbed proteins on PCBMAA-1T polymer brushes is less than 0.8 ng/cm(2) for fibrinogen and 0.3 ng/cm(2) (detection limit of the surface plasmon resonance sensor) for both undiluted blood plasma and serum. It is found that the tertiary amine is favorable to obtain good lactone ring stability in switchable PCB materials. Titration study showed that PCBMAA-1T could resist pH changes under both acidic (pH 1-3) and neutral/basic (pH 7-9) conditions. To the best of our knowledge, such an all-in-one material has not been reported. We believe this material might be potentially used for a variety of applications, including tissue engineering, chronic wound healing and medical device coating.

A microfluidic Coulter counting device for metal wear detection in lubrication oil.

We present the design, fabrication, and testing of a microfluidic device for metal wear detection in lubrication oils. The detection is based on the capacitance Coulter counting principle, that is, on the change in a microchannel's capacitance caused by the presence of a metal particle in the microchannel. The testing of the microfluidic device using 10-25 microm aluminum particles has demonstrated the feasibility for detection and counting of microscale metal particles in low-conductive lubrication oils. This microfluidic device is promising for online oil debris detection by the use of multiple detection microfluidic channels.