Label-free High-throughput Detection Research Articles

Label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters by linear-array-based photoacoustic tomography.

Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification <italic<in vivo</italic<. The feasibility was first demonstrated by imaging CTC cluster <italic<ex vivo</italic<. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats <italic<in vivo</italic<. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters <italic<in vivo</italic< and showed its potential for tumor metastasis study and cancer therapy.

Label-free high-throughput detection and content sensing of individual droplets in microfluidic systems

This study reports a microwave-microfluidics integrated approach capable of performing droplet detection at high-throughput as well as content sensing of individual droplets without chemical or physical intrusion. The sensing system consists of a custom microwave circuitry and a spiral-shaped microwave resonator that is integrated with microfluidic chips where droplets are generated. The microwave circuitry is very cost effective by using off-the-shelf components only. It eliminates the need for bulky benchtop equipment, and provides a compact, rapid and sensitive tool compatible for Lab-on-a-Chip (LOC) platforms. To evaluate the resonator's sensing capability, it was first applied to differentiate between single-phase fluids which are aqueous solutions with different concentrations of glucose and potassium chloride respectively by measuring its reflection coefficient as a function of frequency. The minimum concentration assessed was 0.001 g ml(-1) for potassium chloride and 0.01 g ml(-1) for glucose. In the droplet detection experiments, it is demonstrated that the microwave sensor is able to detect droplets generated at as high throughput as 3.33 kHz. Around two million droplets were counted over a period of ten minutes without any missing. For droplet sensing experiments, pairs of droplets that were encapsulated with biological materials were generated alternatively in a double T-junction configuration and clearly identified by the microwave sensor. The sensed biological materials include fetal bovine serum, penicillin antibiotic mixture, milk (2% mf) and d-(+)-glucose. This system has significant advantages over optical detection methods in terms of its cost, size and compatibility with LOC settings and also presents significant improvements over other electrical-based detection techniques in terms of its sensitivity and throughput.

Resonant waveguide grating for monitoring biomolecular interactions.

Label-free detection technologies have been widely used to characterize biomolecular interactions without having to label the target molecules. These technologies exhibit considerable potential in facilitating assay development and enabling new integrated readouts. When combined with high-throughput capability, label-free detection may be applied to small molecule screens for drug candidates. Based on the resonant waveguide grating biosensors, a label-free high-throughput detection system, the Epic(®) System, has been applied to monitor molecular interactions. Here we describe a generic label-free assay to quantitatively measure phospho-specific interactions between a trafficking signal-phosphorylated SWTY peptide and 14-3-3 proteins or anti-phosphopeptide antibodies. Compared with the solution-based fluorescence anisotropy assay, our results support that the high-throughput resonant waveguide grating biosensor system has shown the capability not only for high-throughput characterization of binding rank and affinity but also for the exploration of potential interacting kinases for the substrates. Hence, it provides a new generic HTS platform for phospho-detection.

Label-free high-throughput and real-time detections of protein interactions by oblique-incidence reflectivity difference method

Selected Mouse IgG of 1 mg/mL as target was fabricated on microarray for 500 sample dots. Label-free and real-time reaction dynamic processes were detected between the microarrays with Goat Anti-mouse IgG of 0.02 mg/mL using the oblique-incidence reflectivity difference (OIRD) method. We obtained the reaction results and the reaction dynamic curves of 500 protein dots. In addition, we also used label-free detection of protein microarrays of 10080 sample dots, including BSA and different concentrations of Mouse IgG and Rabbit IgG, by OIRD. The obtained reaction results between the protein microarray with 1 mg/mL Goat Anti-mouse IgG and 1 mg/mL Goat Anti-rabbit IgG are reported herein. Experimental results show that OIRD can be not only label-free high-throughput detection method for biological microarrays but also label-free real-time detection in the interaction processes of biomolecules.

Systems biology coupled with label-free high-throughput detection as a novel approach for diagnosis of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a treatable and preventable disease state, characterised by progressive airflow limitation that is not fully reversible. Although COPD is primarily a disease of the lungs there is now an appreciation that many of the manifestations of disease are outside the lung, leading to the notion that COPD is a systemic disease. Currently, diagnosis of COPD relies on largely descriptive measures to enable classification, such as symptoms and lung function. Here the limitations of existing diagnostic strategies of COPD are discussed and systems biology approaches to diagnosis that build upon current molecular knowledge of the disease are described. These approaches rely on new 'label-free' sensing technologies, such as high-throughput surface plasmon resonance (SPR), that we also describe.

Oblique-incidence reflectivity difference microscope for label-free high-throughput detection of biochemical reactions in a microarray format

We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in. x 3 in. (2.54 cm x 7.62 cm) microscope slide.