TITLE: Wireless electrochemical measurement of kanamycin in whole blood
AUTHORS: Jun-Chau Chien, Alexandra E. Rangel, Hyongsok T. Soh

[DESCRIPTION]
This work evaluates the life-time of the structural-switching aptamer sensors in the whole blood using in vitro blood circulation setup as the first step toward implantable usage. The device consists of a 3-electrode sensing probe with the tip of the working electrode (WE) functionalized with the antibiotics aptamers. Square-wave voltammetry (SWV) is used to continuously track both the signal peak and the background current at 5 sec temporal resolution. The sensing probe features a diameter of 800um and is inserted through a 18G catheter. Two samples of whole blood (EDTA treated), spiked with either 500uM kanamycin or SSC 1x (salin sodium citrate) buffer, are alternated toward the sensor to evaluate the degradation of the aptamer sensivitiy and the degree of drift.

[STEPS]
SECTION: Aptamer Immobilization
2. Use heat-shrink and bare gold wire (250 um) to define the sensing area at the wire tip.
SECTION: Aptamer Immobilization
3. Solvent clean with sonication in acetone, methanol, and DI-water.
SECTION: Aptamer Immobilization
4. Perform electrochemical cleaning using 3-wire electrochemical system 
a. 3 CV cycles in 200mM EC4 H2SO4
b. 3 CV cycles in 50mM EC3 H2SO4
CV parameters: -0.4V ~ 1.5V, step = 0.001V, rate = 0.001V/sec
SECTION: Aptamer Immobilization
7. Pipette 2 µL TCEP solution in to an aptamer aliquot (1 µLat 100 micromolar concentration, stored at -20 °C upon receiving in dry format) and incubate for 40 min in dark conditions. 






SECTION: Aptamer Immobilization
8. Add 97 µL of 1X SSC buffer into the aliquot after 40 min time period.


SECTION: Aptamer Immobilization
9. Immerse the gold wire into the TCEP-aptamer-SSC solution, incubate for 60 min in dark conditions.

SECTION: Aptamer Immobilization
10. After 60 min take out the gold wire, rinse with DI-water for 1 min and immerse in DI-water for 2 min



SECTION: Aptamer Immobilization
11. Prepare C6-solution: 1.245 mL of DI-water + 1 µL of C6 solution.


SECTION: Aptamer Immobilization
12. Immerse the gold wire into the C6-solution, incubate for 120 min.

SECTION: Aptamer Immobilization
13. After 2 hours, rinse the gold wire with DI-water for 1 min, and immerse in DI-water for 2 min.


SECTION: Aptamer Immobilization
14. Store the gold wire in 1X SSC buffer at 4 °C overnight (1440 min) to stabilize the formation of the self-assembly monolayer (SAM).


SECTION: Aptamer real-time sensor measurement
15. Blood circulation loop (Figure 1 and Figure 2) is created with pump and tubing to emulate actual condition.

 
 

SECTION: Aptamer real-time sensor measurement
16. Human whole blood sample (9 mL, stored at4 °C upon receiving) is split into two separate reservoir, each with 4.5 mL. In the first reservoir, 0.5 mLof 5mM kanamycin in SSC buffer is added. The final concentration is therefore 500uM. In the second reservoir, 0.5mL of SSC buffer without kanamycin is added. Each reservoir has two opening for connecting to the two ports of the circulation loop.




SECTION: Aptamer real-time sensor measurement
17. Insert the sensing probe and connect to the electronics.
SECTION: Aptamer real-time sensor measurement
19. PalmSense software is used to perform square-wave voltammetry (SWV) continuously. The parameters are listed below. Two SWV sweeps are performed sequentially at two different frequencies. The results are subtracted to mitigate the drift.
 

SECTION: Aptamer real-time sensor measurement
20. After each sweep, the measured current is stored in .CSV file. The results are post-processed in Matlab to extract: (1) background current, and (2) signal current, as shown in Figure 3. 
 

SECTION: Aptamer real-time sensor measurement
18.  After the insertion of the aptamer probe, the pump is activated to circulate the blood. A 30 min stabilization period is needed. During this period, the potential are swept based on the parameters below. 
 
 SWV frequency # of steps Potential step size Start potential End potential SWV amplitude 1st SWV sweep 400 Hz 400 1mV -0.4 V 0 V 36 mV 2ndSWV sweep 60 Hz 80 5mV -0.4 V 0 V 36 mV 
 
SECTION: Aptamer real-time sensor measurement
21. After 30-min stabilization period, the two samples are alternated using valves. Due to difference in the antibiotics concentration, the measurements resemble a square wave. The rise and fall time indicates the achievable temporal resolution, which is currently governed by the diffusion rate of the target across the porous membrane. Figure 4 shows an example of the measured results

 

SECTION: Aptamer Immobilization
5. Prepare 100 micromolar stock of kanamycin aptamer (5'-HS-(CH2)6-GGGACTTGGTTTAGGTAATGAGTCCC-(CH2)7-NH-MB-3’). Aptamer is functionalized with a thiol and methylene blue on the 5' and 3' termini respectively.
SECTION: Aptamer sensor probe overview
1. The aptamer-immobilized sensing probe will be approximately 6 ~ 10 cm in length and ~1mm in diameter in order to fit into the 18G catheter. The sensing probe consists of 3 wires: 
(1)  A working electrode (WE) made of a gold wire (Au, Alfa Aesar Inc.) with its tip functionalized with aptamers at approximately 2 mm in length, as defined by the heat shrink tubing (McMaster Carr Inc.). The diameter of the gold wire is approximately 250um. 
(2)  A reference electrode (RE) made of silver chloride (AgCl, A-M Systems) with a diameter approximately 100 um (by immersing silver wires in bleach overnight). 
(3)  A counter electrode (CE) made of platinum (Pt, A-M Systems). 
SECTION: Aptamer Immobilization
6. Prepare fresh TCEP solution at a concentration of 28.7 mg/mL in water.