Ultrasensitive electrocatalytic detection of COX-2 rs20417: relying on 3D interconnected architecture of Pt-LSSUs@PAA nanostructures for sensor interface modification

Figures & data

Scheme 1. (A): The stepwise synthesise procedure of Pt-LSSUs@PAA. (B) Schematic illustration of the electrochemical DNA bioassay protocol.

Table 1. Synthetic oligonucleotide sequences in this study.

Nucleotide name	Sequence (5′→3′)
PNA	NH2-Cys-O-GGC-TGT-ATA-TCT-GCT-CTA-TAT-GC
Target DNA	GCA-TAT-AGA-GCA-GAT-ATA-CAG-CC
Mismatch-1	GCA-TAT-ACA-GCA-GAT-ATA-CAG-CC
Mismatch-2	GCA-TAT-ACA-GCA-GAT-AAA-CAG-CC
Mismatch-3	GCA-TAT-ACA-GGA-GAT-AAA-CAG-CC
Non-complementary	GAG-CAT-GCG-ATC-ACG-CCG-GCT-AG

Figure 1. (A) Typical FE-SEM and (B) TEM images of Pt-LSSUs@PAA nanostructures. (C) EDS of Pt-LSSUs@PAA nanosturctures. (D) EDX elemental mapping of Pt-LSSUs@PAA nanosturctures.

Figure 2. (A) CVs of (a) bare GCE, (b) GCE/Pt, and (c) GCE/Pt-LSSUs@PAA in 5 mM [Fe(CN)₆]^3−/4− containing a 0.1 M KCl solution. (B) Cyclic voltammograms of the modified electrode before (curve b) and after (curve c) hybridisation with target DNA in 5 mM [Fe(CN)₆]^3−/4− containing a 0.1 M KCl solution. (C) EIS for each immobilisation step in 5 mM [Fe(CN)₆] ^3−/4− containing a 0.1 M KCl solution: (a) bare GCE, (b) GCE/Pt-LSSUs@PAA, (c) GCE/Pt-LSSUs@PAA/PNA probe, (d) GCE/Pt-LSSUs@PAA/PNA probe/MCH (e) GCE/Pt-LSSUs@PAA/PNA probe/MCH/target DNA in 5 mM [Fe(CN)₆]^3−/4− containing a 0.1 M KCl solution.

Figure 3. Effects of (A) the concentration of Pt-LSSUs@PAA, (B) immobilisation time of PNA probe on the modified electrode and (C) incubation time.

Figure 4. (A) DPV signals of the DNA sensors in the presence of different concentrations of COX-2 and (B) the calibration curve of the DNA sensors for 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM of COX-2 (n = 3). (C) Specific response of the DNA biosensor spiked with 50 nM glucose, 50 nM urea acid (UA), 50 nM ascorbic acid (AA), and after the biosensor was hybridised with complementary COX-2 (target DNA), single-base mismatch (Mismatch-1, wild type), two-base mismatch (mismatch-2), three-base mismatch (mismatch-3), non-complementary target (random) and blank sample (100 pM for each analyse). (D) The stability of the COX-2 DNA sensors (100 pM) after 3, 7, 10, 14, 21 and 28 days.

Table 2. Reproducibility of the electrochemical detection of COX-2.

COX-2 (pM)		0.01	1	100
RSD (%)	Intra-assay	3.45	3.02	3.15
	Inter-assay	4.01	3.87	3.99

Table 3. Recovery of COX-2 in human serum samples.

Samples	Added	Found*	Recovery (%)	RSD (%)
Sample-1	0.1 pM	0.088 pM	88.00	3.678
Sample-2	1 pM	0.999 pM	99.90	2.202
Sample-3	100 pM	103.090 pM	103.09	2.897

Samples (1–3) were from healthy people.

* The values shown here are the average values from five measurements.

Table 4. Assay results of COX-2 gene in real serum sample that using the DNA sensor and the standard method.

Samples	Sequencing	The DNA sensor	Current (μA)*	△Current (μA)	Calculated concentration (pM)
S1-S10	−	−	−	−	−
S11	+	+	−8.169	−6.243	1.47
S12	+	+	−9.027	−7.101	11.30
S13	+	+	−9.816	−7.890	92.24
S14	+	+	−8.483	−6.557	1.86
S15	+	+	−9.974	−8.048	102.63
S16	+	+	−8.049	−6.123	1.15
S17	+	+	−8.400	−6.474	1.68
S18	+	+	−9.724	−7.797	84.59

Note: – represents the negative results of the COX-2 type, + represents the positive result of the COX-2 type.

* Calculated as a mean of five measurements.