Christian D. Ahrberg

Polymerase chain reaction in microfluidic devices

The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.

Palm-Sized Device for Point-of-Care Ebola Detection

We show the utilization of a recently developed cellphone-sized real-time polymerase chain reaction (PCR) device to detect Ebola virus RNA using single-step reverse transcription PCR (RT-PCR). The device was shown to concurrently perform four PCRs, each with a sample volume of 100 nL: one positive control with both Ebola and GAPDH RNA and one negative control. The last two positions were used to measure the GAPDH and the Ebola content of a sample. A comparison of threshold cycles (CT) from the two samples provided relative quantification. The entire process, which consisted of reverse transcription, PCR amplification, and melting curve analysis (MCA), was conducted in less than 37 min. The next step will be integration with a sample preparation unit to form an integrated sample-to-answer system for point-of-care infectious disease diagnostics.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

Analysis of 3D multi‐layer microfluidic gradient generator

We developed a three‐dimensional (3D) simple multi‐layer microfluidic gradient generator to create molecular gradients on the centimeter scale with a wide range of flow rates. To create the concentration gradients, a main channel (MC) was orthogonally intersected with vertical side microchannel (SC) in a 3D multi‐layer microfluidic device. Through sequential dilution from the SC, a spatial gradient was generated in the MC. Two theoretical models were created to assist in the design of the 3D multi‐layer microfluidic gradient generator and to compare its performance against a two‐dimensional equivalent. A first mass balance model was used to predict the steady‐state concentrations reached, while a second computational fluid dynamic model was employed to predict spatial development of the gradient by considering convective as well as diffusive mass transport. Furthermore, the theoretical simulations were verified through experiments to create molecular gradients in a 3D multi‐layer microfluidic gradient generator.

Poisson statistics-mediated particle/cell counting in microwell arrays

Precise determination of particle or cell numbers is of importance for a wide array of applications in environmental studies, medical and biological applications, or manufacturing and monitoring applications in industrial production processes. A number of techniques ranging from manual counting to sophisticated equipment (e.g., flow cytometry) are available for this task. However, these methods are either labour intensive, prone to error, or require expensive equipment. Here, we present a fast, simple method for determining the number density of cells or microparticles using a microwell array. We analyze the light transmission of the microwells and categorize the microwells into two groups. As particles/cells contained in a microwell locally reduce the light transmission, these wells displayed a lower average transmission compared to unoccupied microwells. The number density of particles/cells can be calculated by Poisson statistics from the ratio of occupied to unoccupied microwells. Following this approach, the number densities of two different types of microparticles, as well as HeLa and E. Coli cells, ranging over four orders of magnitude were determined. Through the microwell array defined by microfabrication, a simple image recognition algorithm can be used with the formation of aggregates or irregular shaped samples providing no additional difficulty to the microwell recognition. Additionally, this method can be carried out using only simple equipment and data analysis automated by a computer program.

Doubling Throughput of a Real-Time PCR

The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.

Functional Graphene Oxide-Based Nanosheets for Photothermal Therapy

Cancer is one of the main causes of morbidity and mortality. Although a number of techniques are available for treatment, these methods still have a number of drawbacks, destroying healthy tissues and cells to cause various side effects. Here we present the synthesis and biological application of a composite nanomaterial, folic acid (FA)-conjugated graphene oxide (GO) nanosheets functionalized with manganese dioxide (MnO2) nanoparticles. While FA-conjugated GO nanosheets can be used for targeted photothermal therapy (PTT) when irradiated with a near infrared (NIR) light, MnO2 nanoparticles degrade hydrogen peroxide (H2O2) in the cancer microenvironment, countering hypoxia. Further the nanoparticles can be used as a contrast agent in MRI imaging. We demonstrated that MnO2-FA-GO nanosheets were uptaken by HeLa cells overexpressing FA receptors to induce NIR irradiation-mediated hyperthermia (35% viability). Therefore, this composite MnO2-FA-GO nanosheet could be a powerful carrier for cancer targeting and PTT applications.

Superheated droplets for protein thermal stability analyses of GFP, BSA and Taq-polymerase

Here we describe a novel method for the study of protein thermal stability using superheated aqueous samples within virtual reaction chambers. Virtual reaction chambers consist of an aqueous sample droplet encapsulated by an oil droplet on a hydrophobic surface. Such samples can be superheated due to the lack of nucleation sites. The thermal denaturation of proteins is induced through the application of a temperature gradient using a bespoke silicon heating chip. The unfolding of proteins is followed through the addition of a hydrophobic dye that attaches to protein hydrophobic domains that become exposed during denaturation. Using this method, we investigated the thermal stability of green fluorescence protein and Taq-polymerase. A possible screening application of the method was demonstrated by evaluating the effect of ionic concentration on the thermal stability of bovine serum albumin.

Dual-nozzle microfluidic droplet generator

The droplet-generating microfluidics has become an important technique for a variety of applications ranging from single cell analysis to nanoparticle synthesis. Although there are a large number of methods for generating and experimenting with droplets on microfluidic devices, the dispensing of droplets from these microfluidic devices is a challenge due to aggregation and merging of droplets at the interface of microfluidic devices. Here, we present a microfluidic dual-nozzle device for the generation and dispensing of uniform-sized droplets. The first nozzle of the microfluidic device is used for the generation of the droplets, while the second nozzle can accelerate the droplets and increase the spacing between them, allowing for facile dispensing of droplets. Computational fluid dynamic simulations were conducted to optimize the design parameters of the microfluidic device.

Circular-shaped microfluidic device to study the effect of shear stress on cellular orientation

Understanding the effects of shear stress on mammalian cells is a crucial factor for understanding a number of biological processes and diseases. Here, we show the development of a circular‐shaped microfluidic device for the facile generation of shear stress gradients. With this microfluidic device, the effect of shear stress on orientation of human umbilical vein endothelial cells was studied. This microfluidic device, which enables to control the alignment of human umbilical vein endothelial cells within a microchannel, can be a valuable tool to mimic blood vessels.