In this section, we demonstrate the preliminary result of biosensing using SiSPR sensor chip. To begin with, Tro4 aptamer42 is modified SiSPR chips and the reactions are monitored via the P-S phasogram. We will use diffusion limited Langmuir model to have a closer look at the surface modification condition. Finally, we will introduce our preliminary results on the cardiac troponin I detection.
“Aptamer” refers to a short strand DNA that has a functionality of antibody to a specific
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protein. Due to the room temperature stability, ease of synthesis and the potential for further chemical structure amendment, aptamer has been widely applied as probe molecule in detection of Platelet-Derived Growth Factor (PDGF)25, interferon gamma for Tuberculosis screening16 and human chorionic gonadotropin detection15.
In 2015, Hunho Jo et. al.42 has reported a series of Aptamer obtained by “Systematic Evolution of Ligands by Exponential enrichment” (SELEX)43 for detection of Cardia Troponin I (cTnI). cTnI is considered as a gold standard biomarker for screening of Acute
Myocardial Infraction (AMI)44. Based on report from Hunho Jo et. al., they have reported a “Tro4” 40 mer aptamer sequence that has a dissociation constant (Kd) of 270 pM for
cTnI monomer and has a Kd of 3.10 nM even for Troponin Complex. In the reported electrochemical sensing experiment, the aptamer can reach a limit of detection around 1 pM (S/N=3), which is lower than clinical cut-off value of 70-400 pg/mL. This suggest a high clinical value of such screening in case of AMI. Since AMI is an indication that suits the targeted application of the portable diagnostics, surface modification of Tro4 aptamer for cTnI detection is selected as a demonstration of bio-sensing efficacy in present work.
The sequence of the Tro4 is 5’-TTT TTT CGT GCA GTA CGC CAA CCT TTC TCA TGC GCT GCC CCT CTT A-3’ (46 mer). The synthesis of the sequence is carried out by
PurigoTM in Taiwan. The 5’ end of the Tro4 aptamer is modified with a thiol functional
group for reaction with SiSPR gold surface. The running buffer for the sensing process is
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1X TE buffer (10 mM Tris + 1 mM EDTA from Protech Technology Enterprise) with 1
M of NaCl. Thiolated Tro4 is firstly mixed with 20 mM of TCEP
(Tris(2-Carboxyethyl)phosphine hydrochloride) for reduction of disulfide bond. The purpose of
the reduction is to confer consistent surface modification efficiency and it typical stand
for more than one hour.
As shown in Fig. 53, before surface modification, a calibration step curve is firstly established by four reference solutions. The calibration step starts with running buffer as baseline, followed by 0.5 % glucose solution, 1 % glucose solution and 1.5 % glucose
Fig. 53 Tro4 Aptamer surface modification
A calibration is first carried out with four reference solutions, followed in influx of Tro4 aptamer. After ~4000 seconds of surface modification, running buffer is flowed again to take confirm the binding of the aptamer.
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solution respectivelyk. The existence of calibration steps helps to transfer phase information into refractive index unit and allows fair comparison of surface modification performance between different chips. After calibration steps, running buffer is used again to re-establish the baseline. The First black arrow in the graph indicates the influx of the
Tro4 aptameric probe. The brown trace indicates the inflow of 250 nM Tro4 DNA while the red and blue trace indicate the influx of 1 M sample. Finally, running buffer is flown
again to cast away the non-specifically bound aptamer as well as to confirm the stringency of probe binding to the plasmonic layer. By blue and red trace, we can see that the modification results are reproducible between different chip trial run.
There are some points that worth discussion before we continue to explore the cTnI detection results. First, judging on papers from Peterlinz et. al.45 and from Peterson et. al.
46, ∆n for ssDNA modification is on order of 10-3~10-2 RIUl, which correspond to our
results here. To be more specific, Peterlinz et. al. conducted the surface modification of 25 mer DNA in 1 M KH2PO4 (pH~3) while Peterson et. al. concluded that 1 M KH2PO4 has more less the same surface modification performance as TE buffer with 1 M NaCl.
Therefore, we can say that, in terms of surface chemistry, we have similar condition as the work from Peterlinz. Under condition mentioned above, Peterlinz’s surface modification lead to a ∆n of 0.0052 RIU for thiolated ssDNA. Considering the fact that
k Glucose reference solutions are prepared in TE running buffer background
l Assuming that DNA has similar refractive index between 650 nm and 850 nm.
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Tro4 aptamer that we use herein has 46 codon, which should have 1.84 times more refractive index change as compared to the sequence used by Peterlinz et. al., ∆n for our case should be somewhere below 0.00968 . To further analyze surface modification data, we have conducted a curve fitting using diffusion limited Langmuir model:
∆n = 𝑛𝑚𝑎𝑥(1 − 𝑒−𝑘𝑡
1 2)
The results is shown in the inset of Fig. 53. The fitting indicates that for surface modification of Tro4 with 2 M concentration, the fitting gives:
∆n = 0.0029(1 − 𝑒−0.038𝑡
1
2) with R2 of 0.96 And for Tro4 modification with 250 nM, the fitting gives
∆n = 0.0014(1 − 𝑒−0.040𝑡
1
2) with R2 of 0.96
For both cases, the maximum amount of surface modification (𝑛𝑚𝑎𝑥) is smaller than 0.00968. The difference may partly due to the stem-loop structure 42of the Tro4 aptamer which requires more free space for ease of steric effect.
Fig. 54 black trace demonstrates the results for cTnI detection using SiSPR sensor chips. As in case of Tro4 Modification, a calibration step is flown for transferring phase information into refractive index unit. The running buffer of cTnI is 1X TE buffer with 150 mM of NaCl. As indicated in the insets, the prominent sensing signal is revealed at 120 nM of cTnI concentration. Considering the fact that molecular weight of cTnI is
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similar to Tro4 aptamerm, we can directly use ∆n𝑐𝑇𝑛𝐼 vs ∆n𝑇𝑟𝑜4 to calculate the binding efficiency. Based on the fact ∆n𝑇𝑟𝑜4 (the red trace in the figure) is 0.001375 when 5000 s of surface modification is done and ∆n𝑐𝑇𝑛𝐼 is around 0.0001 as shown in the inset, we conclude that that There is around 7% of surface Tro4 bound of cTnI at 120 nM. This value is much lower judging from the reported 270 pM KD42
. With a dissociation constant of 270 pM, based on chemical kinect, we can derive that:
270 𝑝𝑀 = [𝐴] × 120𝑛𝑀
In other words, 99.78 % of Tro4 should be complex with cTnI as compared to what we have measured. We considered that the difference in binding performance is due to buffer background. In the reports from Jo. et. al.42, they uses PBS with 10 mM of NaCl, 5 mM of KCl and 1 mM of MgCl2. Apparently, the Tro4 in such condition has much more effective charge (longer Debye length). Moreover, Tro4 would have more stable secondary structure due to the presence of Magnesium ion and lack of EDTA. Therefore, in the future work, we would substantial change the buffer background to reach the maximum performance of the sensor.
m cTnI molecular weight is around 24 kDa. The DNA has around 500 D on average for a single base pair.
In other words, Tro4 has a molecular weight of 46*500~23 kDa.
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