Chengxun Liu

Cell manipulation with magnetic particles toward microfluidic cytometry

Magnetic particles have become a promising tool for nearly all major lab-on-a-chip (LOC) applications, from sample capturing, purification, enrichment, transport to detection. For biological applications, the use of magnetic particles is especially well established for immunomagnetic separation. There is a great amount of interest in the automation of cell sorting and counting with magnetic particles in LOC platforms. So far, despite great efforts, only few fully functional LOC devices have been described and further integration is necessary. In this review, we will describe the physics of magnetic cell sorting and counting in LOC formats with a special focus on recent progress in the field.

Manipulation of magnetic particles on chip by magnetophoretic actuation and dielectrophoretic levitation

The prospect of using magnetic particles for biomedical purposes in lab-on-a-chip systems compels accurate and flexible particle manipulation. Toward such a goal we designed a microdevice comprising a pair of meander-shaped current carrying conductors, which enable simultaneous magnetophoresis and dielectrophoresis by generating both a traveling magnetic field and an ac electric field. Therefore, both the in-plane and out-of-plane movements of magnetic particles can be electrically controlled. A transport speed of tens of μm∕s was achieved with actuation forces at piconewton scale. The enhanced control of particle movement avoids the contact and nonspecific adhesion between the particle and device.

On-chip separation of magnetic particles with different magnetophoretic mobilities

Recent integrations of giant magnetoresistive sensor into laboratory-on-a-chip systems enable the direct detection of biological entities such as cells coated with magnetic particles on chip. However, before detection the different biological entities need to be separated. As a model system, we investigated the separation of two types of magnetic particles (4.5 and 2μm in diameter). The motion of the particles was studied when actuated using an alternating traveling magnetic field produced by four-phase conductors on chip. Different magnetic particles migrate with different speeds in the same traveling magnetic field. By carefully choosing the frequency of the magnetic field, different magnetic particles can be separated in a microfluidic system.

Discrimination of specific and non-specific bindings by dielectrophoretic repulsion in on-chip magnetic bio-assays.

Affinity binding is the principle used in a large number of bio-assays. Aside from specific bindings, non-specific bindings usually deteriorate assays by giving false positive signals and restrict the detection limit. Currently, the assay specificity is mainly dependent on the effectiveness of a suitable surface chemistry. We report an approach to discriminate specific and non-specific bindings with dielectrophoretic (DEP) forces for on-chip magnetic bio-assays. Conjugated to the analytes, magnetic particles were used as the agents for DEP force generation. Due to a weaker binding strength, the non-specifically bound particles were removed while specific bindings remained intact. Analytical and finite element calculations were also performed to study all relevant forces. Furthermore, the removal of magnetic particles was also assessed by measuring the magnetic signal using magnetoresistive sensors. This technique can not only be used to improve the specificity of the on-chip bio-assays but also be developed as a tool of force spectroscopy for the study of bio-molecular binding physics.

Single cell viability observation in cell dielectrophoretic trapping on a microchip

We reported a microfluidic integrated dielectrophoretic (DEP) device for single MCF-7 cell trapping and studied the different effects of applied electric field on the viability of the trapped cell. The cell remained alive when DEP voltage was 3 V. Above 3 V, cell viability significantly decreased when increasing stimulation time. At 8 V, the cell was rapidly lysed by the electric field. The high transmembrane potential induced was found to be the major cause of cell damage. The obtained results indicated that an operational electric field below than 2 kV/cm was safe for cell viability when using DEP for cell manipulation.

Automated genotyping of circulating tumor cells

Cancer remains a prominent health concern in modern societies. Continuous innovations and introduction of new technologies are essential to level or reduce current healthcare spending. A diagnostic platform to detect circulating tumor cells (CTCs) in peripheral blood may be most promising in this respect. CTCs have been proposed as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. Still, their detection remains a challenge as they may occur at concentrations below one single cell per ml of blood. To facilitate their detection, here we describe microfluidic modules to isolate and genotype CTCs directly from clinical blood samples. In a first cell isolation and detection module, the CTCs are immunomagnetically enriched, separated and counted. In a second module and after cell lysis, the mRNA is reversely transcripted to cDNA, followed by a multiplex ligation probe amplification of 20 specific genetic markers and two control fragments. Following the multiplex ligation probe amplification reaction, the amplified fragments are electrochemically detected in a third and final module. Besides the design of the modules, their functionality is described using control samples. Further testing using clinical samples and integration of all modules in a single, fully automated smart miniaturized system will enable minimal invasive testing for frequent detection and characterization of CTCs.

Real-time PCR to study the sequence specific magnetic purification of DNA

The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non‐specific adsorption of DNA/RNA. However, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real‐time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification efficiency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations.

Specific magnetic isolation for direct detection of HPV16

Finding a suitable DNA purification system is vital for the success of many PCR based diagnostic tests. This report demonstrates the value of magnetic beads in combination with real-time PCR for the sequence-specific isolation and detection of episomal HPV16 DNA. In order to maximize the isolation, two purification procedures were evaluated. Compared to the indirect method, in which the target was magnetically labeled after being hybridized to the capture probes, much higher efficiencies were obtained by directly capturing the target using DNA functionalized beads. These higher efficiencies were obtained by carefully tuning the capture probe density on the beads. When modifying the beads with dual-biotinylated capture probes or introducing beads modified with different capture probes, the amount of HPV16 isolated from spiked clinical swab samples even increased further. This not only resulted in the use of dual-biotinylated capture probes in higher purification efficiencies, but also the thermostability of the DNA-bead linkage was found to improve. In summary, this study shows that DNA functionalized magnetic beads are very promising diagnostic tools as they allow for a specific, simple, and fast isolation and concentration of minute quantities of DNA from complex clinical samples.

High throughput cell sorter based on lensfree imaging of cells

Introduction: The trend towards digitization of healthcare promises a future of improved, insight driven, more economical and patient-centered care models. Besides digitization of health records, it has spurred massive research in nano- and bioMEMS devices that sense health related parameters in a compact format. A myriad of devices including innovative CMOS FET structures, nanophotonic and nanomechanical structures, nanowires and nanopores[1,2,3] are being explored as suitable sensors for bio-molecules in ultralow concentrations. Large scale integration offered by silicon technology becomes indispensable when analyzing large amounts of different bio-entities. The most compelling example is the sequencing of DNA with CMOS based platforms. Millions of sensing sites are required to cover the informative genetic aberrations of the human genome, that in total contains 3 billion base pairs. Silicon CMOS technology has been the enabling technology[4,5] to reduce the cost of analysis to a few 100 USD per analysis. Equal emphasis should be put on scaling the fluidic networks required to prepare samples towards those molecular detection devices in order to speed up the total analysis time starting directly from patient material. Figure 1 shows such a silicon microfluidic chip[6], to detect single nucleotide polymorphisms (SNPs), those SNP aberrations are important e.g. for analyzing cancer subtypes, drug response prediction.

Fabrication of silicon based microfluidics device for cell sorting application

In this paper we report on a novel silicon fabrication process of a microfluidics device used for cell sorting application. The process uses wafer level fabrication technique that allows for a small form factor device in combination with a new photopatternable polymer with excellent properties for microfluidics on silicon. The ability to process on wafer level distinguish this from other processes, whose yields are limited to few test samples. The device fabrication includes: processing of micro-heaters, definition of polymer microfluidic channels and collective die-to-wafer bonding of glass substrate onto the polymer channels. The paper will focus on the development of a wafer scale process flow and initial characterization of the available devices.