Negative Enrichment of Circulating Tumor Cells in Blood Using a Microfluidic Chip.

The enumeration and analysis of circulating tumor cells (CTCs) is an increasing interest for monitoring disease progression or response to treatment, specifically as a companion diagnostic for new anticancer drugs, and for research into the mechanisms of disease progression and metastases. Ideally, CTCs would be enriched from very small samples, with minimal handling, high recovery, and no requirement for the expression of specific surface markers. Here, we describe negative enrichment as the preferred approach for cancer cell isolation using a microfluidic platform.

An integrated on-chip platform for negative enrichment of tumour cells.

The study of cancer cells in blood, popularly called circulating tumour cells (CTCs), has exceptional prospects for cancer risk assessment and analysis. Separation and enrichment of CTCs by size-based methods suffer from a well-known recovery/purity trade-off while methods targeting certain specific surface proteins can lead to risk of losing CTCs due to Epithelial to Mesenchymal Transition (EMT) and thus adversely affect the separation efficiency. A negative selection approach is thus preferred for tumour cell isolation as it does not depend on biomarker expression or defines their physical property as the separation criteria. In this work, we developed a microfluidic chip to isolate CTCs from whole blood samples without targeting any tumour specific antigen. This chip employs a two-stage cell separation: firstly, magnetophoresis depletes the white blood cells (WBCs) from a whole blood sample and is then followed by a micro-slit membrane that enables depleting the red blood cells (RBCs) and retaining only the tumour cells. By creating strong magnetic field gradients along with customized antibody complexes to target WBCs, we are able to remove >99.9% of WBCs from 1:1 diluted blood at a sample processing rate of 500µL/min. This approach achieves an average of >80% recovery of spiked tumour cells from 2mL of whole blood in a total assay processing time of 50min without multiple processing steps.

Microfluidic immunomagnetic cell separation from whole blood.

Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells.

Dopamine-assisted deposition of dextran for nonfouling applications.

Nonfouling surfaces are essential for many biomedical applications, such as diagnostic biosensors and blood- or tissue-contacting implants. In this study, we demonstrate a simple one-step method to introduce dextran onto various substrates based on dopamine polymerization. It has been shown for the first time that dextran molecules could be incorporated into a dopamine polymerization product via mixing dextran with dopamine in a slightly alkaline solution. The codeposited film was characterized by X-ray photoelectron spectroscopy (XPS), the water contact angle, ellipsometry, and atomic force microscopy (AFM). Results reveal that it is possible to control the thickness and surface roughness via the deposition time and deposition repeat cycles. Furthermore, quartz crystal microbalance (QCM) measurements show that the dextran-modified surface inhibits protein adhesion. In addition, cell attachment has been significantly inhibited on dextran-modified surfaces even after exposure to water for as long as 2 months. The described dopamine-assisted dextran modification represents a simple and universal method for nonfouling surface preparation and can be potentially applied to improve the performance of various medical devices and materials.

Microfluidic platform for negative enrichment of circulating tumor cells.

Negative enrichment is the preferred approach for tumor cell isolation as it does not rely on biomarker expression. However, size-based negative enrichment methods suffer from well-known recovery/purity trade-off. Non-size based methods have a number of processing steps that lead to compounded cell loss due to extensive sample processing and handling which result in a low recovery efficiency. We present a method that performs negative enrichment in two steps from 2 ml of whole blood in a total assay processing time of 60 min. This negative enrichment method employs upstream immunomagnetic depletion to deplete CD45-positive WBCs followed by a microfabricated filter membrane to perform chemical-free RBC depletion and target cells isolation. Experiments of spiking two cell lines, MCF-7 and NCI-H1975, in the whole blood show an average of >90 % cell recovery over a range of spiked cell numbers. We also successfully recovered circulating tumor cells from 15 cancer patient samples.

Lab-on-a-chip technology: impacting non-invasive prenatal diagnostics (NIPD) through miniaturisation.

This paper aims to provide a concise review of non-invasive prenatal diagnostics (NIPD) to the lab-on-a-chip and microfluidics community. Having a market of over one billion dollars to explore and a plethora of applications, NIPD requires greater attention from microfluidics researchers. In this review, a complete overview of conventional diagnostic procedures including invasive as well as non-invasive (fetal cells and cell-free fetal DNA) types are discussed. Special focus is given to reviewing the recent and past microfluidic approaches to NIPD, as well as various commercial entities in NIPD. This review concludes with future challenges and ethical considerations of the field.

Towards an optimal and unbiased approach for tumor cell isolation.

Our current understanding of clinical significance or the lack thereof of circulating tumor cells (CTCs) is biased by the technology used to isolate these rare cells. Despite the presence of a vast number of academic and commercial technologies, the lack of a standardized and optimized platform has been widely noted. We present a negative enrichment approach, integrating WBC depletion and chemical-free RBC depletion in the same setup without the need for centrifugation, washing or multiple sample handling steps. This approach achieves an average of >90 % recovery of spiked tumor cells and >99 % total WBC depletion in whole blood across multiple cell lines, in a simple and easy-to-use assay. The results presented herein and ongoing improvements aim to fulfill the need for a highly reliable, unbiased, standardized, and optimized CTC isolation platform, using component technologies that are validated for cell isolation.

Effects of metal layer morphology to silicon nanostructure formation in metal-assisted etching.

This study presents a rapid and simple approach for creating silicon nanostructures using metal-assisted etching. The thickness of the metal layer was found to be a key process parameter affecting the surface morphology of silicon nanostructures. Au and Ag layers with a thickness of 3 nm, 5 nm, and 10 nm were used to study the effects of metal catalyst thickness on silicon nanostructure morphology. The experimental results show that the surface morphology of metal has a significant influence on the silicon nanostructure morphology, such that the silicon nanostructures transform from porous silicon surfaces into filament nanostructures or silicon nanowire with increasing thicknesses of both the Au and Ag metal layers.

Compact optical microfluidic uric acid analysis system.

We designed, fabricated and tested a novel compact fluorescence analysis system for quantification of uric acid (UA) in clinical samples at the point-of-care. To perform an analysis, diluted saliva, urine or blood samples are simply placed in a disposable thin-film sample holder using a dropper. A new enzyme immobilization technique was developed to retain within the sample holder two enzymes and a molecule, which transforms into a fluorescer in amounts depending on the UA concentration. The small instrument (7.5 cm × 5 cm × 5 cm) into which the sample holder is placed for analysis contains an LED, a narrow-band filter and an amplified photodiode. The analysis time is 30s, and the dynamic range of the system is 4-400 µM of UA. The calibration curve for transparent saliva and urine was made using solutions of UA. The calibration curve for opaque blood was obtained with spiked samples of blood. The three different types of clinical samples were collected from three subjects and simply diluted before their measurements. Analysis with our instrument yielded UA concentrations within the expected concentration ranges. Development of instruments based on the current laboratory prototype is expected to result in products for clinical trials and point-of-care.

Computational methodology for absolute calibration curves for microfluidic optical analyses.

Optical fluorescence and absorption are two of the primary techniques used for analytical microfluidics. We provide a thorough yet tractable method for computing the performance of diverse optical micro-analytical systems. Sample sizes range from nano- to many micro-liters and concentrations from nano- to milli-molar. Equations are provided to trace quantitatively the flow of the fundamental entities, namely photons and electrons, and the conversion of energy from the source, through optical components, samples and spectral-selective components, to the detectors and beyond. The equations permit facile computations of calibration curves that relate the concentrations or numbers of molecules measured to the absolute signals from the system. This methodology provides the basis for both detailed understanding and improved design of microfluidic optical analytical systems. It saves prototype turn-around time, and is much simpler and faster to use than ray tracing programs. Over two thousand spreadsheet computations were performed during this study. We found that some design variations produce higher signal levels and, for constant noise levels, lower minimum detection limits. Improvements of more than a factor of 1,000 were realized.