The broad availability of smartphones has provided new opportunities to develop less expensive, portable, and integrated point-of-care (POC) platforms. Here, a platform that consists of three main components is introduced: a portable housing, a centrifugal microfluidic disc, and a mobile phone. The mobile phone supplies the electrical power and serves as an analysing system. The low-cost housing made from cardboard serves as a platform to conduct tests. The electrical energy stored in mobile phones was demonstrated to be adequate for spinning a centrifugal disc up to 3000 revolutions per minute (RPM), a rotation speed suitable for majority of centrifugal microfluidics-based assays. For controlling the rotational speed, a combination of magnetic and acoustic tachometry using embedded sensors of the mobile phone was used. Experimentally, the smartphone-based tachometry was proven to be comparable with a standard laser-based tachometer. As a proof of concept, two applications were demonstrated using the portable platform: a colorimetric sandwich immunoassay to detect interleukin-2 (IL-2) having a limit of detection (LOD) of 65.17 ng/mL and a fully automated measurement of hematocrit level integrating blood-plasma separation, imaging, and image analysis that takes less than 5 mins to complete. The low-cost platform weighing less than 150 g and operated by a mobile phone has the potential to meet the REASSURED criteria for advanced diagnostics in resource limited settings.

To date, blood plasma analytes are frequently being used as accepted biomarkers for disease [1]. In order to efficiently detect these analytes, the sample is preferred to be completely free of cells. The lack of a generic integrated blood plasma separation technique has long been one of the obstructions for the large-scale adaptation of microfluidic-based sample-to-answer diagnostic assays, especially for larger sample volumes. Here, we present a robust, scalable, low-cost and secure technology that automatically measures, and dispenses sequentially and proportionally a given volume of blood plasma inside a centrifuge tube.

The simultaneous flow of gas and liquids in large scale conduits is an established approach to enhance the performance of different working systems under critical conditions. On the microscale, the use of gas-liquid flows is challenging due to the dominance of surface tension forces. Here, we present a technique to generate common gas-liquid flows on a centrifugal microfluidic platform. It consists of a spiral microchannel and specific micro features that allow for temporal and local control of stratified and slug flow regimes. We investigate several critical parameters that induce different gas-liquid flows and cause the transition between stratified and slug flows. We have analytically derived formulations that are compared with our experimental results to deliver a general guideline for designing specific gas-liquid flows. As an application of the gas-liquid flows in enhancing microfluidic systems' performance, we show the acceleration of the cell growth of E. coli bacteria in comparison to traditional culturing methods.

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.

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 growing popularity of smartphones has been allowing new opportunities towards the development of low-cost and integrated point-of-care diagnostic platforms. Here, we combine (1) the capabilities of smartphones as both imaging devices and power sources; (2) centrifugal microfluidic devices and; (3) our recently reported microdispenser technology allowing reagent storage, dispensing and blood-plasma separation, to pave the way towards cost-effective and portable point-of-care devices with potential to meet the ASSURED criteria outlined by the World Health Organization.

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

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/μl. Copyright 

Anyone carrying a smartphone has the potential access to an electrical power bank capable of spinning a lab-disc, a sensor for imaging and the processing power to analyze data. Here, exploit the electrical power stored in smartphones to introduce a smartphone operated centrifugal platform made of cardboard and a small motor to provide a low-cost, portable, sample-to-answer centrifugal diagnostic systems, specifically designed to meet the needs in resource-limited-settings. As a proof of principle, sandwich ELISA immunoassay for detection of interleukin-2 and hematocrit level measurement are demonstrated. 

The flow of liquids in centrifugal microfluidics is unidirectional and dominated by centrifugal and Coriolis forces (i.e., effective only at T-junctions). Developing mechanisms and discovering efficient techniques to propel liquids in any direction other than the direction of the centrifugal force has been the subject of a large number of studies. The capillary force attained by specific surface treatments, pneumatic energy, active and passive flow reciprocation and Euler force have been previously introduced in order to manipulate the liquid flow and push it against the centrifugal force. Here, as a new method, the moment of inertia of the liquid inside a chamber in a centrifugal microfluidic platform is employed to manipulate the flow and propel the liquid passively towards the disc center. Furthermore, the effect of the moment of inertia on the liquid in a rectangular chamber is evaluated, both in theory and experiments, and the optimum geometry is defined. As an application of the introduced method, the moment of inertia of the liquid is used in order to mix two different dyed deionized (DI) waters; the mixing efficiency is evaluated and compared to similar mixing techniques. The results show the potential of the presented method for pumping liquids radially inward with relatively high flow rates (up to 23 mm3/s) and also efficient mixing in centrifugal microfluidic platforms.