The success of lab-on-a-chip systems may depend on a low-cost device that incorporates on-chip storage and fluidic operations. To date many different methods have been developed that cope separately with on-chip storage and fluidic operations e. g., hydrophobic and capillary valves pneumatic pumping and blister storage packages. The blister packages seem difficult to miniaturize and none of the existing liquid handling techniques despite their variety are capable of proportional repeatable dispensing. We report here on an inexpensive robust and scalable micro-dispenser that incorporates long-term storage and aliquoting of reagents on different microfluidics platforms. It provides long-term shelf-life for different liquids enables precise dispensing on lab-on-a-disc platforms and less accurate but proportional dispensing when operated by finger pressure. Based on this technology we introduce a method for automation of blood plasma separation and multi-step bioassay procedures. This micro-dispenser intends to facilitate affordable portable diagnostic devices and accelerate the commercialization of lab-on-a-chip devices.

There is a growing recognition of the importance of point-of-care tests (POCTs) for detecting critical neonatal illnesses to reduce the mortality rate in newborns, especially in low-income countries, which account for 98 percent of reported neonatal deaths. Lactate dehydrogenase (LDH) is a marker of cellular damage as a result of hypoxia-ischemia in affected organs. Here, we describe and test a POC LDH test direct from whole blood to provide early indication of serious illness in the neonate. The sample-inresult- out POC platform is specifically designed to meet the needs at resource-limited settings. Plasma is separated from whole blood on filter paper with dried-down reagents for colorimetric reaction, combined with software for analysis using a smartphone. The method was clinically tested in newborns in two different settings. In a clinical cohort of newborns of Stockholm (n = 62) and Hanoi (n = 26), the value of R using Pearson's correlation test was 0.91 (p < 0.01) and the R-2 = 0.83 between the two methods. The mean LDH (+/- SD) for the reference method vs. the POC-LDH was 551 (+/- 280) U/L and 552 (+/- 249) U/L respectively, indicating the clinical value of LDH values measured in minutes with the POC was comparable with standardized laboratory analyses.

Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (mu CAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng mu l(-1). Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng mu l(-1), which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.

The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (similar to 8 nL capture chamber) coupled with a thin-film photodiode (200 x 200 mu m area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 mu L of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.

Point-of-Care (POC) diagnostics often fail to meet the market requirements of low cost and advanced functionality, and are often limited to lateral flow based serological diagnostics with reduced sensitivity and specificity. We report here on an integrated microfluidic absorbance measurement device fabricated by roll-to-roll (R2R) compatible manufacturing processes, suitable for low cost POC systems. It is a device exclusively made of foils and takes external light from a low cost LED and converts the point light source to a homogeneous light via a foil based optical filter at the bottom of the device. The light is converted to an electrical signal by an amorphous organic semiconductor (OSC) material, integrated with screen-printed carbon finger on top of the device for electrical measurement. As a proof of principle, we demonstrate DNA hybridization assay, where the target DNA is coupled to magnetic beads for absorbance measurement. The device successfully distinguishes between matched and mismatched DNA hybridization and can differentiate between 1 μM, 50 nM and 2.5 nM DNA target concentrations. The inherent characteristics of the substrates and R2R fabrication concept significantly reduce the cost, making it suitable for POC applications at resource-limited settings. 

Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.

There is a growing demand for simple, affordable, reliable and quality-assured point-of-care (POC) diagnostics for use in resource-limited settings. Among the top ten leading causes of death worldwide, three are infectious diseases, namely, respiratory infections, HIV/AIDS and diarrheal diseases (World Health Organization 2012). Although high-quality diagnostic tests are available, these are often not available to patients in developing countries. While recent development in microfluidics and “lab-on-a-chip” devices has the potential to spur the development of protocols and affordable instruments for diagnosis of infectious disease at POC, integration of complex sample preparation and detection into automated molecular and cellular systems remain a bottleneck for implementation of these systems at resource-limited settings. Towards this, we describe here how low-cost optical drives can, with minor modifications, be turned into POC diagnostic platforms. A DVD drive is essentially a highly advanced and low-cost optical laser-scanning microscope, with the capability to deliver high-resolution images for biological applications. Furthermore, the inherent centrifugal force on rotational discs is elegantly used for sample preparation and integration. Hence, the merging of low-cost optical disc drives with centrifugal microfluidics is feasible concept for POC diagnostics, specifically designed to meet the needs at resource-limited settings.

We report for the first time the MicroCAP technique, for rapid DNA detection and quantification, that does not require any purification or fluorescent labelling of DNA. The invention is based on DNA interacting with a detection dye (Gelred) to form a complex, that forms a visible precipitate within seconds of centrifugation. MicroCAP can be used for DNA quantification, when combined with the Lab-on-DVD with inbuilt centrifugation and sub- micron imaging resolution. We quantify PCR and LAMP assay products using MicroCAP on the integrated Lab-on- DVD platform, and demonstrate a detection limit of 10 ng/!".

KEYWORDS: MicroCAP, DNA detection, Centrifuge,Precipitate, LAMP, PCR.

INTRODUCTION

Detection of amplified DNA is often based on measurement of turbidity, fluorescence (after staining with a detec- tion dye) or absorbance. Commercially available instruments for DNA quantitation can be broadly divided into two categories: UV instruments based on absorbance (such as spectrophotometers, e.g. Nanodrop or Nanophotometer) and instruments based on measurement of a fluorescent dye (such as plate readers). One bottleneck in quantifying amplified DNA in a nucleic acid amplification test (NAAT) reaction, based on absorbance measurement technique, is the bias introduced due to the presence of the isothermal amplification buffer, dNTPs and other reagents. Each reagent or buffer may have an absorbance density at around 260 nm, elevating the apparent concentration measured by the device compared to the actual value. Hence, for most quantitation based NAATs, it is important to include an extra DNA purification step, which may result in non-negligible loss of the amplified product and increases the cost of the purification kit. Measurements based on fluorescence mostly use fluorescent dyes that are potentially hazardous for handling. In addition, fluorescence based quantitation methods require time consuming labelling and washing steps.

In this report, we describe a new method, termed microfluidic centrifugation assisted precipitation (microCAP), involving quantification and detection of DNA based on precipitation of nucleic acids. The basis of the method is formation of a visible precipitate when GelRed, a nucleic acid intercalacting dye commonly used in gel electropho- resis, is mixed with DNA and centrifuged. A visible precipitate is formed after just a few seconds of centrifugation and enables rapid detection of the presence of DNA in a sample. To the best of our knowledge, the visible precipitate formed as a product of centrifuging GelRed mixed with DNA has not been reported before. We showed that the DNA GelRed complex is dense enough compared to water to precipitate upon centrifugation. Further, we extended the μCAP method to the Lab-on-DVD platform1 to quantify the DNA concentration from images generated using the optical DVD reader instrument. The modified DVD player was able to image the precipitate formed up to a detection limit of 10 ng/μl of DNA. For calibration of the images, known quantities of a purified PCR product were used to identify the relationship between the amounts of DNA and precipitate formed. We applied the method to quantify an unknown quantity of LAMP amplicons from a LAMP assay for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. A sensitivity limit of 10 ng/μl of DNA was achieved, comparable with that of a Nanophotometer.18 The results demonstrated that the method is able to quantitatively detect the presence of DNA in a sample in a few seconds without any purification step.

EXPERIMENTAL

The Lab-on-DVD system was employed for spinning and imaging the precipitate product using a modified DVD drive, as mentioned in our previous report.1 We began by dispensing the sample in the design chamber, adding GelRed dye (at a concentration of 4000X in water) and centrifuging the mixture at 1200 rpm. Figure 1a and 1b

show schematics of the DNA sample precipitation process conducted in test tubes and the DVD platform, respec- tively. We used known amounts of a PCR product to calibrate the quantity of precipitate to the DNA concentration. We used a HIV genome amplified from 50 ng of plasmid pNL4.3 using the primers 0776F and 6231R.2 To evaluate the sensitivity of DNA detection of our system, we used the amplified products from a LAMP assay. The sensitivity of LAMP primers was tested on DNA from pNL4.3 (a HIV-1B genome containing plasmid). A 25X LAMP primer mix was prepared according to Curtis et al.,3 using the same template DNA sequence, set of primers and DNA polymerase. Eight concentrations (each being 5 μl volume) of the HIV-1B genome containing plasmid (pNL4.3) were tested, starting from 1 ng/!" serially diluted to 1 fg/!". Two negative controls were also prepared, one without DNA and primers and one without primers. The total reaction volume was increased to 30 μl (instead of 25 μl used in Curtis et al.3) by multiplying every component volume in the reaction by a factor of 1.2. Fabrication of the multi- layer microfluidic Disc followed the same procedure as described in our previous report.1 The Lab-on-DVD system was used to generate images of the precipitation zone. To quantify the amount of precipitate, an image processing script was written in MATLAB software (Mathworks, USA).

RESULTS AND DISCUSSION

MicroCAP was found to be suitable for determining the presence of DNA in a sample, We carried out the LAMP assay in Eppendorf tubes in an oven set at 65°C. After 45 minutes, 3 μl of 10,000X GelRed in water was added to two tubes of 30 μl volume each, one having an unknown concentration of LAMP amplified DNA and the other one with no DNA template as a control. After centrifugation for approximately 5 seconds, a visible precipitate was formed in the tube containing amplified DNA, whereas no precipitate was formed in the control tube (Fig. 2a). 10 μl volume of DNA was inserted into a U shaped channel of the DVD alongwith 1 μl of 10,000X GelRed in water, which was the same ratio of DNA sample to Gelred as used in the test tube. An imageable precipitate was observed in the Lab on DVD custom imaging software (fig.2b).

A Matlab script was used for image analysis in which an original image(fig.3a) was transformed into a binary image (fig.3b) by defining a threshold pixel value, exploiting the difference in intensity of the precipitate from its background. The entire area to the left of the threshold line in the histogram (Fig. 3c), i.e. from value 0 to the threshold value (normally 90), was summed to estimate the total area of the precipitate.

For DNA quantification, known concentrations of a PCR product was used for calibration. The initial concentration of purified PCR product was 129 ng/μl, measured with a Nanophotometer (in triplicates) after purification with a GeneJet PCR purification kit. The purified PCR product was subsequently diluted serially several times and each diluted concentration was measured again with the Nanophotometer (in triplicate). The measurements were then repeated with the Lab-on-DVD method. Fig. 4a shows four images recorded at four known concentrations together with their binary threshold images. Fig. 4b shows the precipitation area calculated from the images plotted against the known DNA concentrations, showing a linear relationship. 10 ng/μl was the lowest concentration detectable in the DVD images.

For quantification of unknown quantities of nucleic acids, we carried out the LAMP assay on HIV-1B genome containing plasmid DNA using serial dilutions (10-fold dilutions from 1 ng/μl to 0.1 fg/μl) to evaluate the limit of detection (Fig.5). Two negative controls were also prepared, one comprising primers and no DNA template and second, no DNA template and no primers.

Fig. 6 shows the precipitation area plotted against the starting concentration of DNA template. It shows that the amplification in the LAMP assay is not linear for all the starting concentrations of DNA template. The error bars in the figure show the standard deviation for a particular concentration. For a LAMP assay, which fluctuates somewhat in its yield of amplified prod- ucts, we believe that this error range is acceptable.

The precipitation area was converted to an actual yield of DNA products for each of the concentrations. This conversion was based on the linear empirical equation generated from the calibration curve presented earlier in Fig. 4b, given by:

y= 9.61x – 4.05 (1) Here, y denotes the precipitation area in arbitrary units while x denotes the DNA concentration.

CONCLUSION

We demonstrated an extremely fast visual DNA quantification method (μCAP) that can be made quantifiable on a Lab-on-DVD platform. The approach was based on DNA forming a precipitate upon centrifugation when in contact with the GelRed dye. Results using HIV-1B genome containing plasmid DNA revealed a detection limit of 0.01 pg/μl or total amount of 0.1 pg of starting DNA template, which is an acceptable standard for resource limited settings. The limit of detection of DNA with the Lab-on-DVD platform was found to be 10 ng/μl, which is almost comparable to the detection limits reported by commercially available instruments, such as the Nanophotometer. However, the μCAP method offers a distinct advantage over other state-of-the-art techniques as it does not require additional purification of the DNA. We believe the μCAP technique combined with the Lab-on-DVD platform provides a simple and low cost technology that can fulfil the need for a point-of-care device for DNA quantification.

REFERENCES

1. [1]  H. Ramachandraiah, M. Amasia, J. Cole, P. Sheard, S. Pickhaver, C. Walker, V. Wirta, P. Lexow, R. Lione and A. Russom, "Lab-on-DVD: standard DVD drives as a novel laser scanning microscope for image based point of care diagnostics."Lab. Chip, 2013, 13, 1578–1585.

2. [2]  S. Grossmann, P. Nowak, and U. Neogi, “ Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical man- agement.” Journal of the International AIDS Society, 2015,18(1), 20035.

3. [3]  K. A. Curtis, D. L. Rudolph, I. Nejad, J. Singleton, A. Beddoe, B. Weigl, P. LaBarre and S. M. Owen, "Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT- LAMP)." PLoS ONE, , DOI:10.1371/journal.pone.0031432.

CONTACT

*A. Russom; phone: +46-87909863; aman@kth.se

Bioparticles such as mammalian cells and bacteria can be manipulated directly or indirectly for multiple applications such as sample preparation for diagnostic applications mainly up-concentration, enrichment & separation as well as immunoassay development. There are various active and passive microfluidic particle manipulation techniques where Acoustophoresis is a powerful technique showing high cell viability. The use of disposable glass capillaries for acoustophoresis, instead of cleanroom fabricated glass-silicon chip can potentially bring down the cost factor substantially, aiding the realization of this technique for real-world diagnostic devices. Unlike available chips and capillary-based microfluidic devices, we report milliliter-scale platform able to accommodate 1ml of a sample for acoustophoresis based processing on a market available glass capillary. Although it is presented as a generic platform but as a demonstration we have shown that polystyrene suspending medium sample can be processed with trapping efficiency of 87% and the up-concentration factor of 10 times in a flow through manner i.e., at 35µl/min. For stationary volume accommodation, this platform practically offers 50 times more sample handling capacity than most of the microfluidic setups. Furthermore, we have also shown that with diluted blood (0.6%) in a flow-through manner, 82% of the white blood cells (WBCs) per ml could be kept trapped. This milliliter platform could potentially be utilized for assisting in sample preparation, plasma separation as well as a flow-through immunoassay assay development for clinical diagnostic applications.