By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

As an efficient, rapid and label-free micro-/nanoparticle separation technique, dielectrophoresis (DEP) has attracted widespread attention in recent years, especially in the field of biomedicine, which exhibits huge potential in biomedically relevant applications such as disease diagnosis, cancer cell screening, biosensing, and others. DEP technology has been greatly developed recently from the low-flux laboratory level to high-throughput practical applications. In this review, we summarize the recent progress of DEP technology in biomedical applications, including firstly the design of various types and materials of DEP electrode and flow channel, design of input signals, and other improved designs. Then, functional tailoring of DEP systems with endowed specific functions including separation, purification, capture, enrichment and connection of biosamples, as well as the integration of multifunctions, are demonstrated. After that, representative DEP biomedical application examples in aspects of disease detection, drug synthesis and screening, biosensing and cell positioning are presented. Finally, limitations of existing DEP platforms on biomedical application are discussed, in which emphasis is given to the impact of other electrodynamic effects such as electrophoresis (EP), electroosmosis (EO) and electrothermal (ET) effects on DEP efficiency. This article aims to provide new ideas for the design of novel DEP micro-/nanoplatforms with desirable high throughput toward application in the biomedical community.

On the design, functions, and biomedical applications of high-throughput dielectrophoretic micro-/nanoplatforms: a review.

Sample preparation, as a key procedure in many biochemical protocols, mixes various samples, and/or reagents into solutions that contain the target concentrations. Digital microfluidic biochips (DMFBs) have been adopted as a platform for sample preparation because they provide automatic procedures that require less reactant consumption and reduce human-induced errors. However, the most existing methods only consider two-reactant sample preparation, and they cannot be used for many biochemical applications that involve multiple reactants. In addition, the existing methods that can be used for multiple-reactant sample preparation were proposed on traditional DMFBs where only the (1:1) mixing model is available. In the (1:1) mixing model, only two droplets of the same volume can be mixed at a time, which results in higher completion time and the wastage of valuable reactants. To overcome this limitation, the micro-electrode-dot-array (MEDA) architecture has been introduced; it provides the flexibility of mixing multiple droplets of different volumes in a single operation. In this article, we present a generic multiple-reactant sample preparation algorithm that exploits the novel fluidic operations on MEDA biochips. We also propose an enhanced algorithm that increases the operation-sharing opportunities when multiple target concentrations are needed, and therefore the usage of reactants can be further reduced. The simulated experiments show that the proposed method outperforms existing methods in terms of saving reactant cost, minimizing the number of operations, and reducing the amount of waste.

Multitarget Sample Preparation Using MEDA Biochips

Digital microfluidic biochips (DMFBs) have emerged as a promising platform for DNA sequencing, clinical chemistry, and point-of-care diagnostics. Recent research has shown that DMFBs are susceptible to various types of malicious attacks. Defenses proposed thus far only offer probabilistic guarantees of security due to the limitation of on-chip sensor resources. A micro-electrode-dot-array (MEDA) biochip is a next-generation DMFB that enables the real-time sensing of on-chip droplet locations, which are captured in the form of a droplet-location map. We propose a security mechanism that validates assay execution by reconstructing the sequencing graph (i.e., the assay specification) from the droplet-location maps and comparing it against the golden sequencing graph. We prove that there is a unique (one-to-one) mapping from the set of droplet-location maps (over the duration of the assay) to the set of possible sequencing graphs. Any deviation in the droplet-location maps due to an attack is detected by this countermeasure because the resulting derived sequencing graph is not isomorphic to the original sequencing graph. We highlight the strength of the security mechanism by simulating attacks on real-life bioassays. We also address the concern that the proposed mechanism may raise false alarms when some fluidic operations are executed on MEDA biochips. To avoid such false alarms, we propose an enhanced sensing technique that provides fine-grained sensing for the security mechanism.

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Secure Assay Execution on MEDA Biochips to Thwart Attacks Using Real-Time Sensing

A digital microfluidic biochip (DMB) is an attractive platform for automating laboratory procedures in microbiology. To overcome the problem of cross-contamination due to fouling of the electrode surface in traditional DMBs, a contactless liquid-handling biochip technology, referred to as acoustofluidics, has recently been proposed. A major challenge in operating this platform is the need for a control signal of frequency 24 MHz and voltage range $\pm 10/\pm 20$ V to activate the IDT units in the biochip. In this paper, we present a hardware design that can efficiently activate/de-activated each IDT, and can fully automate an bio-protocol. We also present a fault-tolerant synthesis technique that allows us to automatically map biomolecular protocols to acoustofluidic biochips. We develop and experimentally validate a velocity model, and use it to guide co-optimization for operation scheduling, module placement, and droplet routing in the presence of IDT faults. Simulation results demonstrate the effectiveness of the proposed synthesis method. Our results are expected to open new research directions on design automation of digital acoustofluidic biochips.

Hardware Design and Fault-Tolerant Synthesis for Digital Acoustofluidic Biochips

Sample preparation, as a key procedure in many biochemical protocols, mixes various samples and/or reagents into solutions that contain the target concentrations. Digital microfluidic biochips (DMFBs) have been adopted as a platform for sample preparation because they provide automatic procedures that require less reactant consumption and reduce human-induced errors. However, traditional DMFBs only utilize the (1:1) mixing model, i.e., only two droplets of the same volume can be mixed at a time, which results in higher completion time and the wastage of valuable reactants. To overcome this limitation, a next-generation micro-electrode-dot-array (MEDA) architecture that provides flexibility of mixing multiple droplets of different volumes in a single operation was proposed. In this paper, we present a generic multiple-reactant sample preparation algorithm that exploits the novel fluidic operations on MEDA biochips. Simulated experiments show that the proposed method outperforms existing methods in terms of saving reactant cost, minimizing the number of operations, and reducing the amount of waste.

Sample preparation for multiple-reactant bioassays on micro-electrode-dot-array biochips

With the growing number of new biomarkers discovered for clinic diagnosis, there is an emerging need for the development of biosensing device. One important application of the biosensor in disease diagnosis is to detect biomarkers and the biosensor should be sensitive enough to measure the small variation in biomarker concentration. Besides, to support the point-of-care diagnostics that can facilitate emergency diagnosis and disease screening, it is important that the sensing device needs to be portable, user-friendly, and support the rapid biomarker detection. In this work, we propose a rapid portable sensing device by utilizing dielectrophoresis force to accelerate the centralization of the biomarkers and the comparator-based operational amplifier to enlarge small signal response. Furthermore, the testing results demonstrate the proposed biosensor can detect subtle admittance variation corresponding to the concentration change of the biomarkers in short assay time. The sensing device is able to detect cardiac troponins level with the limit of detection at 0.1 ng mL−1, that can satisfy the actual requirements for diagnosis of cardiovascular disease. The proposed biosensor can provide an easy-to-use, cost-effective, and rapid biomarker detection that can be applied to clinical applications to support point-of-care diagnostics.

Rapid Portable Electrical Biosensing Design With Dielectrophoresis and Its Application for Cardiac Biomarker Detection

A near-threshold digital LDO (DLDO) with fast transient response is presented in this paper. In order to improve settling time, a voltage-controlled delay line (VCDL) with a new proposed delay cell is developed to enhance conversion gain. In addition, Vernier-delay-line-based time-to-digital convertor (TDC) is used to quantize phase from phase frequency detector (PFD). Furthermore, digital coarse and fine tunings are developed to control PMOS array. The test chip is designed in 90nm SPRVT CMOS and operates at 0.3–0.5V with output voltage of 0.25–0.45V. Simulation results show that current efficiency at 2.8mA is 99.6%. The settling time is only 8us and 12us when load current I LOAD is 200uA and 2.8mA, respectively.

Design of a near-threshold digital LDO with fast transient response

A digital lab-on-CMOS chip is presented for nucleic acid amplification test. This biochip consists of programmable microelectrode dot array and digital timing controller. With novel circuit design in each microelectrode, microfluidic operations with velocity of 0.6mm/s, temperature profile with maximum change rate of 4.5°C/s and location sensing in $\mu $ second-level can be achieved. As a result, test samples can be located first, followed by target operations to meet the requirements of specific medical tests with less reagent consumption and test time/cost. Furthermore bio-protocols can be derived to ensure both replicability and reliability in various medical tests. Preliminary results in loop-mediated isothermal amplification (LAMP) and PCR thermal cycle tests demonstrate the feasibility of our proposal with less test time of < 40 min and < 1 min respectively, making our proposal very suitable for mobile healthcare applications.

Yun-Sheng Chan

Papers

Multitarget Sample Preparation Using MEDA Biochips

Sample preparation for multiple-reactant bioassays on micro-electrode-dot-array biochips

Rapid Portable Electrical Biosensing Design With Dielectrophoresis and Its Application for Cardiac Biomarker Detection

Design of a near-threshold digital LDO with fast transient response

A Programmable Bio-Chip With Adaptive Pattern-Control Micro-Electrode-Dot-Array