Jun 22, 2022

Public workspaceWireless electrochemical measurement of kanamycin in whole blood

DOI
dx.doi.org/10.17504/protocols.io.j8nlk5dydl5r/v1
  • Jun-Chau Chien1,
  • Alexandra E. Rangel1,
  • Hyongsok T. Soh1
  • 1Stanford University
Alexandra Rangel
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk5dydl5r/v1
Protocol Citation: Jun-Chau Chien, Alexandra E. Rangel, Hyongsok T. Soh 2022. Wireless electrochemical measurement of kanamycin in whole blood. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk5dydl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 16, 2019
Last Modified: June 22, 2022
Protocol Integer ID: 19401
Keywords: aptamer, implant, kanamycin, electrochemical
Abstract
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.
Materials
MATERIALS
ReagentKanamycin Monosulfate 50 mg/mL SolutionGold BiotechnologyCatalog #K-120-SL
ReagentTCEP-HClGold BiotechnologyCatalog #TCEP
ReagentHuman whole blood with Na2EDTA
ReagentKanamycin aptamerBiosearch Technologies
Aptamer sensor probe overview
Aptamer sensor probe overview
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).
Aptamer Immobilization
Aptamer Immobilization
Use heat-shrink and bare gold wire (250 um) to define the sensing area at the wire tip.
Solvent clean with sonication in acetone, methanol, and DI-water.
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
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.
Prepare fresh TCEP solution at a concentration of 28.7 mg/mL in water.
Pipette Amount2 µL TCEP solution in to an aptamer aliquot (Amount1 µL at 100 micromolar concentration, stored at Temperature-20 °C upon receiving in dry format) and incubate for Duration00:40:00 in dark conditions.






Add Amount97 µL of 1X SSC buffer into the aliquot after Duration00:40:00 time period.


Immerse the gold wire into the TCEP-aptamer-SSC solution, incubate for Duration01:00:00 in dark conditions.

After Duration01:00:00 take out the gold wire, rinse with DI-water for Duration00:01:00 and immerse in DI-water for Duration00:02:00



Prepare C6-solution: Amount1.245 mL of DI-water + Amount1 µL of C6 solution.


Immerse the gold wire into the C6-solution, incubate for Duration02:00:00 .

After 2 hours, rinse the gold wire with DI-water for Duration00:01:00 , and immerse in DI-water for Duration00:02:00 .


Store the gold wire in 1X SSC buffer at Temperature4 °C overnight (Duration24:00:00 ) to stabilize the formation of the self-assembly monolayer (SAM).


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

Figure 1: Schematic illustration of the blood circulation loop. The sensing probe, with the tip functionalized with aptamer sensors, is inserted into the tube through a 18G catheter. The exposed wires are connected to the potentiostat for electrochemical sensing.
Figure 2: Snapshot of the blood circulation loop setup in the lab.

Human whole blood sample (Amount9 mL , stored atTemperature4 °C upon receiving) is split into two separate reservoir, each with Amount4.5 mL . In the first reservoir, Amount0.5 mL of 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.




Insert the sensing probe and connect to the electronics.
After the insertion of the aptamer probe, the pump is activated to circulate the blood. A Duration00:30:00 stabilization period is needed. During this period, the potential are swept based on the parameters below.
 SWV frequency# of stepsPotential step sizeStart potentialEnd potentialSWV amplitude
1st SWV sweep400 Hz4001mV-0.4 V0 V36 mV
2ndSWV sweep60 Hz805mV-0.4 V0 V36 mV
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.
Note
Parameters are aptamer-specific, i.e. different aptamers will need different sweeping parameters for minimum drift.

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.
Figure3. Example of SWV sweep. The signal of interest is the difference between the peak and the background (or baseline) current.

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

Figure 4: Measured time-domain results.