Experiments

Purpose

To ensure that the aptamer binds to its target biomarker.

Procedure

• Reconstitute the lyophilized aptamer in nuclease-free water to reach a concentration of 100 μM.
• Create the folding buffer using:
  • 1 L DEPC-treated water
  • 8.01 g (137 mmol) NaCl
  • 0.201 g (2.7 mmol) KCl
  • 1.14 g anhydrous disodium phosphate (8 mmol)
  • 0.239 g (1.76 mmol) potassium phosphate
  • 0.1 g (1 mmol) MgCl₂ anhydrous
• Place aptamer in folding buffer, heat to 85–95°C for 5 minutes, then cool to room temperature.
• Mix 1 L of aptamer solution into 100 mL of buffer.
• Aliquot ¼ of the solution into a tube labeled “control”.
• To the remaining solution, add 0.451 g of β-amyloid 42 (1 × 10⁻⁴ moles).
• Incubate and take aliquots as follows:
  • 5 min → tube labeled “5”
  • 10 min → tube labeled “10”
  • 15 min → tube labeled “15”
• Transfer all tubes (control, 5, 10, 15) to wells of a gel electrophoresis apparatus and analyze results:
  • Aptamer not bound to the target travels farther.
  • Aptamer bound to the target travels less far in the gel.

Objective

Attach TMB to the aptamer sequence.

Procedure

• Preparation of Aptamer Sequence:
  • Attach a PEG4-amine to one of the hairpin loops in the DNA sequence.
  • Confirm the presence of PEG4-amine using Poly-Acylamide Gel Electrophoresis (PAGE).
  • Cut the band expected to contain the modified DNA from the gel.
• Preparation of TMB:
  • Synthesize TMB-succinyl.
  • Dissolve 1 mmol TMB in 7.5 mL dry pyridine.
  • Add 1.5 mmol succinic anhydride stock and incubate 4–6 hours at 25°C.
  • Quench by adding 10 mL cold water, adjust pH to 4.5–5 using HCl in 0.5 mL increments.
  • React TMB-succinyl with NHS: Add 1.5 mmol EDC-HCl to MES or DMF buffer, add TMB-succinyl, incubate 3 hours at 25°C.
• Conjugation:
  • Mix activated TMB with 100 μL of 100 μM 3’-amine-modified aptamer in PBS (pH 8.5).
  • Incubate 2 hours at 25°C for linker-aptamer interaction.
  • Quench with Tris-HCl (pH 8.0) for 15 minutes.
  • Remove unreacted TMB via centrifugation at 2500 rpm for 10 minutes using a filtering device.

Objective

Confirm successful function of aptamer-enzyme-substrate complex and its activation when the target is present.

Procedure

Gel Cassette Assembly

• Clean glass plates with detergent, distilled water, then ethanol.
• Assemble the cassette on a flat surface with the shorter plate facing outwards on top of the spacer plate.
• Slide plates into the casting frame, lock in place with clip doors facing outwards.
• Place cassette on gray casting stand gasket, engage spring-loaded lever, and insert pipette tip to ensure tight seal.
• Check for leaks with water and wipe plates dry.

Native PAGE Gel Preparation

• In a 50 mL Falcon tube, mix 2.7 mL 30% acrylamide / 1% bisacrylamide, 1.25 mL 3 M Tris-HCl, bring volume to 10 mL with dH₂O.
• Add 10 μL TEMED and 100 μL 10% ammonium persulfate, mix carefully.
• Pour solution between glass plates using an eyedropper, insert comb at 45° angle.
• Let gel polymerize 20–30 min, remove from casting frame, place in electrode assembly with buffer dam if needed.

Electrophoresis and In-Gel Activity Procedure

• Fill chamber with 1× native running buffer.
• Remove comb carefully.
• Mix 35 μL protein standards or aptamer-enzyme-substrate with 35 μL 2× native loading buffer, incubate 37°C for 15 min.
• Load wells as follows:
  • Lane 1: 20 μL HRP
  • Lane 2: 20 μL aptamer-HRP-substrate
  • Lane 3: 20 μL aptamer only
  • Lanes 4–6: EMPTY
  • Optional: protein ladder to approximate aptamer size
  • Lane 7–9: Repeat as above
• Run gel at 100 V for 60–70 min until tracking dye reaches the bottom.
• Cut gel in half for staining:
  • Lanes 1–5: Coomassie blue staining, rinse, destain overnight, visualize.
  • Lanes 6–10: Incubate with 0.01–0.03% H₂O₂ in PBS for 10–30 min in the dark; blue color indicates HRP activity.

Objective

Confirm functionality of TMSD strands.

Procedure

Gel Cassette Assembly

• Assemble plates in the casting stand.
• Check for leaks by adding water between plates. If none, dump water and wipe dry.

Create Separating Gel (10 mL)

• Prepare the appropriate percentage of acrylamide/bis-acrylamide in a small beaker.
• Add specific volumes of AP and TEMED according to the table below:

Prepare Stacking Gel Solution (5 mL)

• Combine 4.275 mL 0.375M Tris-HCl and 0.57 mL acrylamide/bis-acrylamide in a new beaker.
• Add 0.05 mL 10% AP and 5 μL 1% TEMED, mix gently.
• Pour out water from previous step, fill gaps with stacking gel, insert comb, and let set 20–30 min.

Sample Loading

• Mix sample buffer and prepare sample mixtures for lanes:

Lane	Sample	Purpose
1	DNA ladder	See weights
2	Aptamer	Confirm aptamer weight
3	ONPG	Confirm ONPG weight
4	Aptamer-ONPG	Ensure proper attachment
5	Aptamer-ONPG + invader (T=15)	Control
6	Aptamer-ONPG + invader (T=10)	Test modularity
7	Aptamer-ONPG + invader (T=5)	Test modularity
8	Aptamer-ONPG + invader (T=0)	Test modularity

• Set voltage and run electrophoresis.
• Stain gel to visualize bands.

Objective

Anchoring of the TMSD strand with the nitrocellulose membrane surface to act as the test lane for the LFA.

Procedure

• Incorporate a 5’-biotin onto the 5’ end of the incumbent strand (alpha domain). This will biotinylate the TMSD strand, serving as the receptor region for Streptavidin.
• Prepare 2–5% GMA solution in ethanol/water and incubate the nitrocellulose membrane for 1–2 hours at room temperature.
• Rinse membrane with PBS to remove excess GMA.
• Prepare 1–2 mg/mL streptavidin solution in PBS and add Traut’s reagent (2-iminothiolane) to introduce thiol groups.
• Incubate 1 hour, then purify thiolated streptavidin using a desalting column.
• Streak thiolated streptavidin on the membrane, let dry ~1 hour.
• Dilute biotinylated TMSD-aptamer in PBS, streak over the test line, dry 30–60 minutes to allow conjugation.
• Block membrane by soaking in 1% BSA/PBS for 30–60 minutes, rinse with PBS, and dry at room temperature.

Objective

Discover the pH and temperature ranges at which the sensor is effective and ensure functionality in blood-like conditions.

Procedure

• Test aptamer-TMSD response to biomarker in Tris-HCl at pH 6.0, 7.0, and 8.0.
• Test response at 15°C, 20°C, 25°C, 30°C, and 35°C.
• Place buffer containing known biomarker concentration in the LFA under the specified condition (only one variable at a time).
• Observe biosensor response and measure absorbance at 420 nm.
• Determine concentration from standard curve and calculate percent error: Percent Error = (Obtained Concentration / Actual Concentration) × 100%
• Compare percent error across conditions to assess accuracy and effective operating range.