Team:NYU Abu Dhabi/Documentation/DOCS 20ee279bfcdc46b09c4fb108851b2757/Engineering 8297e0cf88af4f8fbfe00ad598c6a52f/Case Studies b2be0b8bd13548ac99d3f41e1124dbb2/A paper and plastic device for DNA amplification ( b3661fd170db47f896ceb647ff94971e

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• A paper and plastic device for DNA amplification (RPA) + LFA detection + Electrical heating

A paper and plastic device for DNA amplification (RPA) + LFA detection + Electrical heating

A paper and plastic device for performing recombinase polymerase amplification of HIV DNA

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HI

https://pubs.rsc.org/en/content/articlelanding/2012/LC/C2LC40423K

Does not extract DNA, only Amplification + Detection with LFA

Device Setup

Fig 1. Components, assembly, and operation of a paper-based device that performs recombinase polymerase amplification. (A) Devices were constructed from five components: a base layer made of acetate and double-sided adhesive covered by a protective layer of paper; a second acetate layer to aid alignment of other components; a sample wick strip patterned with melted wax for absorbing exactly 10 μL of sample; a cellulose pad for holding a master mix solution; and a glass fiber pad for holding magnesium acetate solution. (B) Devices were assembled by peeling off the protective paper layer and assembling each device component in the order shown. The white pellet shown in the first step is composed of lyophilized enzymes and reagents. (C) The user operates the device by pipetting reagents on the appropriate pads, dipping the wick into the sample, and mixing reaction components by folding the device in half.

Equipment Used + Workflow

• A base layer made of acetate and double-sided adhesive covered by paper, a box is removed from the cover to stick the lyophilized enzymes pellet.

• A cellulose pad for holding the master mix is stuck over the enzyme pellet, a sample wick strip that absorbs exactly 10 microL at its non-wax hydrophilic end is stuck at the end (by exposing the adhesive)

• Another acetate layer is put over the base layer, it has a box hole, in which a glass fiber pad is stuck for holding the magnesium acetate solution for RPA.

• Appropriate reagents are put into the pads, and the sample wick strip is dipped inside the sample solution. The device is then folded to put the different solutions in contact ,see Fig. 1 C

• TwistAmp nfo kit for Lateral flow based detection with the universal strips

• Devices were placed on a heat block at 37 °C for 15 min and covered by a styrofoam box lid for insulation. Devices were then peeled open. The entire sample wick was torn off the device, placed in 90 μL running buffer, and vortexed.

• Ten microliters of diluted products were added to each Milenia lateral flow detection strip, and each strip was placed in a well of a 96-well plate containing 200 μL running buffer. Strips were scanned after 3 min for markings.

Key Findings

• Very low cost amplification platform design that is easy to make as well

• Reagents are either lyophilized or stable at room temperature, hence prevents the need for a cold storage chain

• RPA occurs successfully on paper and requires a shorter incubation time as compared to PCR.

• Cons: Not complete, needs DNA extraction steps, needs pipetting, transferring the products of RPA to the LFA strips is also not a well-defined process