TITLE: Lateral flow buildup
AUTHORS: Jorge Fernández

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following protocol describes how to build a half-strip simplified lateral flow sensor. Preparation of test strips, detection reagents immobilization and test development.</div><div class = "text-block">The purpose of this protocol is testing the proper functioning of the analytical mechanism of a lateral flow assay design. The strips have been simplified for fitting in 96-plate wells. The system doesn't integrate microfluidics for simplification of the assay.</div></div>

[STEPS]
?. [Strips preparation]
Cut 4 pieces of 5mm wide x 40 mm long  strips from the nitrocellulose FF80HP membrane. Label in the plastic backing of the membranes T or C where the Control or Test dots will be deposited. The dots will be positioned at 15  mm and 25 mm from the bottom of the strip. Alternating their order in two of the strips (T/C in two membranes and C/T in the other two).
?. [Strips preparation]
Cut 4 pieces of the cellulose absorbent pad 6mm wide x 12 mm long, and fix them with laboratory paper tape at the top of the strips, overlapping by 6 mm with the membrane. Assure that the tape makes soft pressure, allowing contact between absorbent pad and the membrane.
?. [Reagents preparation]
Pipette on  two different 1.5 mL eppendorf tubes 10 μL of each aptamer suspensions. Put the tubes in a 95 ºC pre-heated thermoblock. Keep the tubes at 95ºC for 10 minutes to allow DNA to denaturalize.
?. [Reagents preparation]
After the 10 minutes, remove the tubes from the thermoblock, and place them quickly on ice. Let them chill for other 10 minutes. That will allow the DNA to acquire the most favorable thermodynamic conformation.
?. [Reagents preparation]
Mix on the previous eppendorf tube 10 μL of 100 μM suspension of amino terminal modified DNA sequence, with 10 μL of the 5 μg/μL  streptavidin suspension. Repeat that step for both DNA sequence to immobilize on the membrane and label the tubes.Incubate the tubes in ice for 1 hour.
?. [Reagents preparation]
Prepare two additional eppendorf tubes and make a 1:6 dilution, taking 2 μL of each Streptavidin-DNA conjugate tubes and diluting in 10 μL of PBS buffer.
?. [Reagents Immobilization]
Place the strips horizontally on a flat surface and label them. A pair of them with different T/C region disposition will hold the 1:6 dilution ( 8 μM) of the reagents, the other one will be treated with the 50 μM prepared suspension.
?. [Reagents Immobilization]
Take 0.33 μL of the correspondant DNA dilution and pipette it very slow on the proper T/C region of the prepared strips. Repeat this step with each one of the four different tubes of streptavidin-biotinylated DNA conjugates.Allow the membranes to dry for aproximately 15 minutes, and repeat three times more the reagents deposition, pipetting exactly in the same point as the previous time. At the end 1.2 μL will have been deposited in each region.
?. [Reagents Immobilization]
After the last deposition of reagents, let the membrane to completely dry, keeping it overnight at 4ºC in a dessicator.
?. [Strip Test]
Mix 200 μL of 1% wt aptamer conjugated beads with 400 μL of E. Coli prepared suspension previously mentioned in required materials. Incubate for 1 hour at room temperature.
?. [Strip Test]
Pipette 125 μL of the E. Coli - Aptamer conjugated beads in in 4 wells of the 96-plate. (125 μL per well, 500 in total).
?. [Strip Test]
Take each previously prepared membrane and fit it into the wells of the 96-plate. Allow sample migration for 30 minutes, letting it to get absorbed into the top absorbent pad.
?. [Reagents Immobilization]
Block the prepared membranes by immersion in 0.2 % BSA suspension for 30 seconds.
?. [Strip Test]
Results can be directly visualized as visible colored spots on the membrane.