| 1. |
- Aljadi, Zenib, et al.
(författare)
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A novel tool for clinical diagnosis of allergy operating a microfluidic immunoaffinity basophil activation test technique
- 2019
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Ingår i: Clinical Immunology. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1521-6616 .- 1521-7035. ; 209
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Tidskriftsartikel (refereegranskat)abstract
- The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel and cumbersome processing, which has limited its clinical use. We therefore investigated if a microfluidic immunoaffinity BAT (miBAT) technique can be a reliable diagnostic method. Blood was collected from allergic patients and healthy controls. Basophils were challenged with negative control, positive control (anti-FccRI), and two concentrations of a relevant and non-relevant allergen. CD203c and CD63 expression was detected by fluorescent microscopy and flow cytometry. In basophils from allergic patients the CD63% was significantly higher after allergen activation as compared to the negative control (p < .0001-p = .0004). Activation with non-relevant allergen showed equivalent CD63% expression as the negative control. Further, the miBAT data were comparable to flow cytometry. Our results demonstrate the capacity of the miBAT technology to measure different degrees of basophil allergen activation by quantifying the CD63% expression on captured basophils.
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| 2. |
- Aljadi, Zenib, et al.
(författare)
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Altered basophil function in patients with chronic kidney disease on hemodialysis
- 2017
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Ingår i: Clinical Nephrology. - : DUSTRI-VERLAG DR KARL FEISTLE. - 0301-0430. ; 88:2, s. 86-96
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Tidskriftsartikel (refereegranskat)abstract
- Aims: Chronic kidney disease (CKD) leads to impairment of immune cell function. Given the potential role of basophils in the pathogenesis of CKD, we aimed to study the basophil responsiveness towards microbial antigen exposure, judged as adhesion molecule expression and degranulation, in CKD patients on hemodialysis. Materials and methods: We selected markers linked to two crucial biological phases: the transmigration and degranulation processes, respectively. For the transmigration process, we selected the adhesion molecules CD11b, active CD11b epitope, and CD62L and for the degranulation process CD203c (piecemeal degranulation marker), CD63 (degranulation marker), and CD300a (inhibitory marker of degranulation). We measured basophil responsiveness after stimulation of different activation pathways in basophils using lipopolysaccharide (LPS), peptidoglycan (PGN), formyl-methyinoyl-leucyl-phenylalanine (fMLP), and anti-FceRI-ab. Results: The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared to matched healthy controls, but no differences were observed after activation by anti-Fc.RI. CD300a expression was significantly higher in patients following activation by fMLP and anti-Fc.RI, and the active epitope CD11b expression was significantly higher in patients after LPS activation. In addition, we found that CD62L was not shed from the cell surface after activation with LPS and fMLP. A slight downregulation was noted after activation with anti-Fc.RI in healthy controls. Conclusion: Together, these data demonstrate that basophil functions related to adhesion and degranulation are altered in CKD patients on hemodialysis, which indicates a potential role for the basophil in the pathogenesis of complications related to infections.
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| 3. |
- Aljadi, Zenib, et al.
(författare)
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Microfluidic Immunoaffinity Basophil Activation Test for Point-of-Care Allergy Diagnosis
- 2019
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Ingår i: Journal of Applied Laboratory Medicine (JALM). - : American Association for Clinical Chemistry. - 2475-7241 .- 2576-9456. ; 4:2, s. 152-163
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Tidskriftsartikel (refereegranskat)abstract
- Background: The flow cytometry-based basophil activation test (BAT) is used for the diagnosis of allergic response. However, flow cytometry is time-consuming, requiring skilled personnel and cumbersome processing, which has limited its use in the clinic. Here, we introduce a novel microfluidic-based immunoaffinity BAT (miBAT) method. Methods: The microfluidic device, coated with anti-CD203c, was designed to capture basophils directly from whole blood. The captured basophils are activated by anti-FceRI antibody followed by optical detection of CD63 expression (degranulation marker). The device was first characterized using a basophil cell line followed by whole blood experiments. Weevaluated the device with ex vivo stimulation of basophils in whole blood from healthy controls and patients with allergies and compared it with flow cytometry. Results: The microfluidic device was capable of capturing basophils directly from whole blood followed by in vitro activation and quantification of CD63 expression. CD63 expression was significantly higher (P = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FceRI-activated captured basophils in microfluidic chip was significantly higher (P = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis (P = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FceRI-activated captured basophils in microfluidic chip compared with flow cytometry. Conclusions: We report on the miBAT. This device is capable of isolating basophils directly from whole blood for on-chip activation and detection. The new miBAT method awaits validation in larger patient populations to assess performance in diagnosis and monitoring of patients with allergies at the point of care.
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| 4. |
- Banerjee, Indradumna, et al.
(författare)
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Analog particle position tuning in Elasto-inertial microfluidic flows
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We observe for the first time an analog trend in particle focusing in a high throughput weakly viscoelastic regime, where it is possible to tune particles into multiple intermediate focusing positions that lie between the "Segre-Silberberg annulus" and the center of a circular microcapillary. The "Segre-Silberberg annulus" (0.6 times the pipe radius), that describes particle equilibrium in a predominantly inertial flow, shrinks consistently closer to the center for increasing elasticity in extremely dilute PEO concentrations (ranging from 0.001 wt% to 0.05wt%). The experimental observations are supported by direct numerical simulations, where an Immersed Boundary Method is used to account for the presence of particles and a FENE-P model is used to simulate the presence of polymers in a Non-Newtonian fluid. The numerical simulations study the dynamics and stability of finite size particles and are further used to analyze particle behavior at Reynolds number higher than what is allowed by the present experimental setup. In particular, we are able to report the entire migration trajectories of the particles as they reach their final equilibrium positions and extend our predictions to other geometries such as the square cross-section. We believe complex effects originate due to a combination of inertia and elasticity in a weakly viscoelastic regime, where neither inertia nor elasticity are able to mask each other's effect completely, thus leading to a number of intermediate focusing positions. The present study provides a new understanding into the mechanism of particle focusing in elasto-inertial flows and opens up new possibilities for exercising analog control in tuning the particle focusing positions.
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| 5. |
- Banerjee, Indradumna, 1986-, et al.
(författare)
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Dynamics of Inertial migration of particles in straight channels
- 2017
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Konferensbidrag (refereegranskat)abstract
- SUMMARYWe study numerically the entire migration dynamics of spherical and oblate particles in straight rectangular and square cross sectional ducts. The reported results can help in design of straight duct channel based microfluidic systems. KEYWORDS: Inertial microfluidics, Lateral migration, Oblate particles, Straight particles. INTRODUCTIONWe simulate spherical and oblate rigid particles in straight ducts of different aspect ratios using an Immersed Boundary Method. To the best of our knowledge, this is the first time not only the equilibrium position of particles is described, but also the entire migration dynamics of the particle from the initial to final position, including particle trajectory, velocity, rotation and orientation, are investigated. EXPERIMENTAL The fluid is considered incompressible and its motion is governed by the Navier Stokes and Continuity equations. The numerical approach employed is an Immersed Boundary Method (IBM) with two sets of grid points: an equispaced Eulerian mesh for the fluid flow, and Lagrangian grid points uniformly distributed on the surface of the particle. The flow is set up in square and rectangular cross section ducts with no slip and no penetration boundary conditions (Fig.1). RESULTS AND DISCUSSIONWe examine the lateral motion of spherical and oblate particles using the IBM method mentioned above. While simulating three different spheres in a square duct of duct width to sphere diameter ratio H/Ds= [3.5, 5, 10], we find that the particles focus at closest face-cantered equilibrium position from their point of introduction(Fig.2a). We also show the downstream length needed for a sphere to focus, focusing length, as a function of the distance from the vertical duct symmetry line and as a function of Reynolds number(Fig.2b and c respectively). Spherical particles in rectangular duct tend to move laterally toward the longer length wall and then slowly moves towards the equilibrium position at the face-centre along the long wall(fig.3a). We also observe that the focusing length is longer for spherical particles in a rectangular duct, about three times longer than that in square duct (fig. 3b). In case of an oblate particle flowing through a square duct, the lateral motion towards the face centred equilibrium position is similar to that of a sphere (fig.4a), however there is significant tumbling motion of the particle as it tries to reach equilibrium(fig.4b).In a rectangular duct of aspect ratio 2, the oblate particle reaches a steady configuration on the duct symmetry line at the center of the different faces (fig.5a). The focusing length surprisingly is shorter in a rectangular duct for an oblate particle in contrast to its focusing length in a square duct. This is attributed to the higher lateral velocity of the oblate in the second stage of the migration, that with negligible tumbling(fig.5b). The behavior of three oblate particles in a square duct of duct width to longer diameter ratio H/Ds= [3.5, 5, 10] is different compared to a sphere as the largest oblate tend to focus at the duct cross section diagonals compared to the other two which are at face centred equilibrium as in case of a sphere(fig.6a). We attribute this to the rotation rate of the larger particle which is initially increasing and then decreasing(fig.6b).When it comes to focusing lengths, the smaller particles need longer times to reach their final equilibrium(fig.6c). Another interesting behavior we see is the effect of Reynolds number, where it can be seen that the oblate particles show a tilt of 21 degrees when focusing at equilibrium at certain high Reynolds number (fig.7). CONCLUSIONThe results presented employ a highly accurate interface-resolved numerical algorithm, based on the Immersed Boundary Method to study the entire inertial migration of an oblate particle in both square and rectangular ducts and compare it with that of a single sphere. Currently, we apply a volume penalization method and polymeric drag component to the code to solve for viscoelastic effects in circular microcapillaries. ACKNOWLEDGEMENTSThis work was supported by the European Research Council Grant no. ERC-2013-CoG-616186, TRITOS and by the Swedish Research Council Grant no. VR 2014-5001, COST Action MP1305: Flowing matter, and computation time from SNIC. REFERENCES : Lashgari, Iman, et al. Journal of Fluid Mechanics 819 (2017): 540-561.
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| 6. |
- Banerjee, Indradumna, et al.
(författare)
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Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics
- 2018. - 2
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Ingår i: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases. - Switzerland : Springer. - 9783319666471 ; , s. 23-38
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Bokkapitel (refereegranskat)abstract
- 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.
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| 7. |
- Banerjee, Indradumna, 1986-, et al.
(författare)
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LDH based neonatal diagnostics on a low-cost slipdisc based sample preparation platform.
- 2016
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Konferensbidrag (refereegranskat)abstract
- INTRODUCTIONSlipdisc is developed as a sample preparation platform based on slipchip technology [1], using a handwinded clockwork mechanism allowing sample processing from one spot to another with defined precision without the need for sophisticated tools or alignment (Fig.1). An ordinary smartphone or camera can be used to image and analyse the results making it an ideal tool for resource limited settings. Here, we demonstrate a bioassay for detecting LDH (Fig.2), a crucial enzyme found in all living cells which leaks out when the cellular membrane is damaged. This makes LDH a biomarker for several medical conditions in newborns, such as Ozkiraz-13, necrotizing enterocolitis (NEC), and Asphyxia.EXPERIMENTALFor assembling the slipdisc optically transparent, robust and disposable CD like polycarbonate discs were used with superhydrophobic coating on all except the embedded microfluidic channels. For the LDH assay, heparinized plasma samples were spiked with 7 different concentrations of the LDH enzyme (Lee Biosolutions, USA). These concentrations ranged from clinically normal to abnormal concentrations and used to construct a standard curve for LDH enzyme.RESULTS AND DISCUSSIONThe ability of the SlipDisc to quantify LDH enzyme levels from plasma samples was evaluated (Fig.3). Using 7 different concentrations, a standard curve with clinically relevant LDH concentrations was obtained (Fig4). Image and data analyses, including linear regression and Pearson’s correlation, were completed using Image processing tool in Matlab.CONCLUSIONWe demonstrate a low-cost neonatal diagnostics platform for the detection of LDH from plasma using a novel SlipDisc platform. The SlipDisc can further be modified to separate plasma from whole blood samples in order to fully integrate the assay. Its simple operation and smartphone based detection capabilities make it an ideal device for point-of-care neonatal diagnostics.
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| 8. |
- Banerjee, Indradumna, et al.
(författare)
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MicroCAP
- 2018
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Patent (populärvet., debatt m.m.)abstract
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| 9. |
- Banerjee, Indradumna, et al.
(författare)
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MicroCap : Microfluidic centrifuge assisted precipitation for DNA quantification on lab-on-DVD
- 2018
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Ingår i: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. - : Chemical and Biological Microsystems Society. - 9781510897571 ; , s. 1802-1805
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Konferensbidrag (refereegranskat)abstract
- 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
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| 10. |
- Banerjee, Indradumna, et al.
(författare)
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MicroCAP: Microfluidic Centrifuge Assisted Precipitation for DNA Quantification on Lab-on-DVD
- 2018
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Konferensbidrag (refereegranskat)abstract
- 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.INTRODUCTIONDetection 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.EXPERIMENTALThe 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 1bshow 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 DISCUSSIONMicroCAP 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.CONCLUSIONWe 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] 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] S. Grossmann, P. Nowak, and U. Neogi, “ Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epide
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