The DNAzyme-Driven Catalytic Hairpin Self-Assembly for the CuNCs Based Electrochemical Detection of HPV16 DNA

Abstract

High-risk human papillomavirus (HPV), particularly HPV16, is a major causative factor in cervical cancer. Its early and highly sensitive detection is crucial for reducing the cancer mortality rates among women. The traditional detection methods suffered from the limitations such as complex procedures and low sensitivity. To solve the issue, this work developed a copper nanoclusters (CuNCs)-based electrochemical biosensor combined with the DNAzyme-driven catalytic hairpin self-assembly (CHA) signal amplification strategy for the highly sensitive detection of HPV16 DNA (deoxyribonucleic acid). In the presence of HPV16 DNA, the duplex formed between a DNA probe and the Cu-substrate was opened, and the Cu-substrate was released. The released Cu-substrate then underwent specific cleavage in the presence of the Cu-enzyme and Cu²⁺, generating two DNA fragments, S1 and S2. The S1 subsequently initiated the CHA cycle, producing a large amount of the duplex formed by two hairpin strands (i.e., H1/H2). The H1/H2 duplex were then bound to the polyadenine (PolyA) probe on the electrode surface, resulting in the in situ synthesis of CuNCs and generating a significantly amplified impedance signal. The results demonstrated that the developed sensor exhibited high sensitivity, good selectivity and satisfactory recovery for the HPV16 DNA detection, which was simple, economical and efficient.

Keywords:
HPV; CHA; DNAenzyme-driven; CuNCs; electrochemcial sensor

Introduction

Human papillomavirus (HPV) is a spherical deoxyribonucleic acid (DNA) virus that is extensively distributed in nature, and humans are its only known host.¹,² This virus primarily targets and infects the epithelial cells of the epidermis and mucosal surfaces, including but not limited to those found in the cervix, anus, oral cavity and various skin regions. More than 200 distinct HPV genotypes have been identified and characterized to date and approximately 15 kinds of HPV genotypes are classified as the high-risk types. These high-risk genotypes have been formally designated as group 1 carcinogens by the International Agency for Research on Cancer, indicating sufficient evidence of their carcinogenicity in humans.²,³ Notably, HPV16 stands out as the most prevalent and highly carcinogenic subtype within this group, which is responsible for nearly 60% of all cervical cancer cases diagnosed across the globe. Cervical cancer is one of the foremost causes of cancer-related mortality among women worldwide.⁴-⁶ This burden is particularly acute in the regions and countries with low and middle income, where the access to the regular and reliable cervical cancer screening programs is often severely limited or unavailable.⁷-⁹ Consequently, the early and highly accurate detection of high-risk HPV infections, with a specific emphasis on identifying HPV16, is of paramount importance.

The traditional methods for HPV detection, such as cytology-based Pap smears, polymerase chain reaction (PCR) and hybrid capture assays, have made significant contributions to clinical screening.¹⁰-¹² However, they still have certain limitations, including complex operating procedures, high requirements for the professional equipment and skilled personnel, long detection cycles and high false-negative rates in the early-stage infection.¹³,¹⁴ Electrochemistry possesses the advantages of sensitivity, simplicity and cost-effectiveness, which has been widely applied in the field of biosensing in recent years.¹⁵,¹⁶ Sabzi et al.¹⁷ previously reported the use of Methylene Blue (MB)-labeled graphite electrodes for the detection of HPV target DNA, utilizing the specific interaction between MB and the guanine base to achieve the signal transduction. Nasirizadeh et al.¹⁸ introduced hematoxylin as an electroactive label for the first time, exploited its differential affinity for the single-stranded and double-stranded DNA, and constructed an electrochemical sensor for HPV DNA detection. This sensor was capable of not only detecting target DNA sequence but also identifying the single-base mismatches. Naorungroj et al.¹⁹ developed an electrochemical sensor integrated into a thermally controlled paper-based digital microfluidic platform. By combining the target-induced hairpin opening with the enzyme-assisted signal amplification, the sensor enabled the rapid detection of HPV16 DNA. These methods mentioned above could detect HPV16 DNA with good performance, but they all required the use of the electroactive labels or expensive enzymes, significantly increasing the cost and hindering their adoption in the resource-limited regions.

Metal nanoclusters (NCs), such as copper nanoclusters (CuNCs), represent an emerging class of nanomaterials. They exhibit numerous unique and superior properties, including distinctive optical and electrical characteristics, exceptional specific surface areas and excellent biocompatibility.²⁰-²² These attributes enable the metal NCs to effectively enhance the electrochemical signal, significantly improving the sensitivity of the sensor. Our group achieved sensitive detection of HPV16 DNA by leveraging the high affinity of polyadenine (PolyA) sequences toward gold nanoparticles in combination with chain displacement or chain-mediated hybridization reactions.²³,²⁴ These studies demonstrated good analytical performance for HPV16 DNA detection. However, they required multiple steps for DNA modification on the electrode surface, which were complex and time-consuming.

Catalytic hairpin self-assembly (CHA) has garnered widespread attention due to its isothermal, high efficiency and excellent biocompatibility.²⁵,²⁶ It achieves effective signal amplification by triggering a cascade hybridization reaction between two hairpin DNA strands under specified conditions, resulting in the formation of abundant duplex DNA.²⁷ As an efficient enzyme-free signal amplification tool, CHA significantly improves the analytical performance of nucleic acid sensing. In addition, DNAzyme are a class of single-stranded DNA molecules with catalytic ability, which could catalyze the cleavage reaction of substrate strands with the assistance of the specific metal ions.²⁸ In recent years, a dual amplification strategy combining DNAzyme with CHA has provided a novel approach to address the challenges in the low-concentration nucleic acid detection. Ren et al.²⁹ integrated CHA with DNAzyme to develop a label-free, ultra-sensitive electrochemical sensor for microRNA detection. Wang et al.³⁰ reported a triple cascade amplification system that ingeniously combined CHA, Mg²⁺-dependent DNAzyme, and hybrid chain reaction (HCR) for ultra-sensitive electrochemical detection of microRNA, achieving a limit of detection as low as 9.25 aM. These studies conclusively demonstrated that the integration of CHA and DNAzyme could generate synergistic amplification effects, providing a viable pathway for precise detection of trace nucleic acid targets. Meanwhile, no work has been reported utilizing the two strategies for HPV DNA detection.

In this work, a CuNCs-based electrochemical sensor with signal amplification by DNAzyme-driven CHA has been developed for the sensitive detection of HPV16 DNA. The detection principle is illustrated in Figure 1. DNA probe a hybridized with the Cu-substrate to form a duplex DNA through the principle of complementary base pairing. When the target was present, HPV16 DNA hybridized with DNA probe a to form a more stable duplex, resulting in the release of the Cu-substrate. The released Cu-substrate then hybridized with the designed Cu-enzyme (a DNA sequence with enzymatic cleavage activity). In the presence of Cu²⁺, the cleavage activity of the Cu-enzyme was activated, cutting the Cu-substrate at the specific site and fragmenting it into two short strands (S1 and S2). The short strand S1 served as the trigger chain for CHA. It first hybridized with probe H1, opening the hairpin to expose its terminal sequence. This terminal sequence hybridized with the stem region of the second hairpin probe H2, opening the H2 hairpin and forming a stable H1/H2 duplex. During this process, short chain S1 was released and catalyzed the next CHA cycle. The resulting H1/H2 duplex hybridized with the terminal region of PolyA probe, which facilitated the in situ synthesis of CuNCs and thus generated a high impedance signal. When HPV16 DNA was absent, the copper substrate could not be released, resulting in the inability of the subsequent DNA enzyme-catalyzed reactions and CHA. As a result, the CuNCs were unable to be synthesized and only the PolyA strand was modified on the electrode, leading to a low impedance signal. The concentration of HPV16 DNA was negatively correlated with the impedance values. The developed sensor performed high sensitivity, good selectivity and satisfactory recoveries for HPV16 DNA detection, providing a new alternative method for the early clinical screening of high-risk HPV infection.

Figure 1
Schematic diagram of the DNAzyme-driven CHA for the CuNCs based electrochemical detection of HPV16 DNA.

Experimental

Chemicals and instruments

HAuCl₄.3H₂O, CuSO₄, sodium ascorbate (SA), 6-mercapto-1-hexanol (MCH), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), ethylenediaminetetraacetic acid (EDTA), and NaCl were purchased from Aladdin-Reagent Company (Shanghai, China). Superior-grade fetal bovine serum was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. (China). The [Fe(CN)₆]^3-/4- solution contained 5 mM K₄[Fe(CN)₆], 5 mM K₃[Fe(CN)₆] and 0.1 M KCl. All solutions were prepared and diluted with ultrapure water obtained from a Milli-Q (Bedford, USA) ultrapure water system.

A conventional three-electrode cell was employed for the electrochemical impedance spectroscopy (EIS) measurements, which were conducted on a CS310H electrochemical workstation (Wuhan Kesite Instruments Co., Ltd., China). In this work, a glassy carbon (GCE) electrode (diameter = 3 mm) was served as the working electrode. A platinum wire was used as the counter electrode and a saturated calomel electrode (SCE) was used as the reference electrode. For the morphological characterization, a Talos F200i transmission electron microscope (Thermo Fisher Scientific, USA) was used to acquire the image.

All the oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology Co., Ltd. (Shanghai, China) and used directly without further purification. Their sequences were as follows:

• - HPV16: 5’-TGCTAACATTGCTGCCTTTGC-3’;

• - HPV18: 5’-GGAATGCTCGAAGGTCGTCT-3’;

• - HPV31: 5’-GGTGAACCGAAAACGGTTGG-3’;

• - HPV51: 5’-TCTGCTGTACAACGCGAAGG-3’;

• - HPV58: 5’-ACAGCTAGGGCACACAATGG-3;

• - PolyA: 5’-AAAAAAAAAAAAAAAAAAAAAAAAA AAAAACACACACACAAATATA-3’;

• - DNA probe a: 5’-GCAAAGGCAGCAACCTTCTCTT GTTAGCA-3’;

• - Cu-substrate: 5’-AGAGAAGGCATAATCTTCGA-3’;

• - Cu-enzyme: 5’-CAAGAATTTTTCTCCGGGTCCTTCT CT-3’;

• - H1: 5’-TCGAAGATTATGCCTATATTAATC-3’;

• - H2: 5’-GATTAATATAGGCATAATCTTCATATATT-3’.

Cu²⁺-dependent DNAzyme-driven CHA

First, 10 μL of DNA probe a (12 μM) and 10 μL of Cu substrate (12 μM) were added to centrifuge tube 1. They were incubated at room temperature for 1 h to achieve complementary base pairing and the formation of a double-stranded structure. Then, 20 μL of HPV16 DNA at a series of concentrations was added to the centrifuge tube and incubated for 1 h. Next, 10 μL of Cu-enzyme (12 μM) was added and incubated for 1 h to obtain the Cu²⁺-mediated DNAzyme structure. After that, 10 μL of CuSO₄ (60 mM) was added to the system and incubated for 1 h to complete the cleavage, yielding the DNA fragments (i.e., S1 and S2). In centrifuge tube 2, 10 μL of H1 solution (1 μM) and 10 μL of H2 solution (10 μM) were added and heated at 90 °C for 20 min, followed by cooling to room temperature and keeping for 30 min. Then, 30 μL of the mixture solution was transferred from centrifuge tube 1 to centrifuge tube 2 and incubated for 1 h to obtain the H1 and H2 duplex.

Electrochemical detection of HPV16 DNA

A mirror-like surface was obtained on the GCE by polishing with 0.05 μm alumina slurry, followed by sequential ultrasonic cleaning in ultrapure water, anhydrous ethanol, and ultrapure water. The polished GCE was then activated in 1 mM H₂SO₄ via cyclic voltammetry between -0.6 and +1.6 V. The PolyA probe exhibited a weak affinity toward the GCE. Thus, it was difficult to immobilize the PolyA probe on the GC electrode without any modification. According to the reported reference,³¹ there was a strong affinity between gold and the PolyA sequence. Therefore, the electrodeposition of gold nanoparticles (AuNPs) onto the GCE surface was used for constructing the electrochemical DNA sensors, which was conventional and economically efficient. The GC electrode was placed in a 5 mM HAuCl₄ solution containing 0.1 M KNO₃ at -0.2 V for 300 s and the obtained electrode was denoted as GCE/Au. Then, 15 μL of PolyA (1 μM) was added to the surface of the GCE/Au electrode and incubated in the dark overnight to form the GCE/Au/PolyA electrode. After rinsing with ultrapure water, the electrode was immersed in 1 mM MCH solution for 30 min to block the non-specific binding sites, obtaining the GCE/Au/PolyA/MCH electrode. Subsequently, 15 μL of the resulting solution obtained by the Cu²⁺-dependent DNAzyme-driven CHA was pipetted onto the surface of the GCE/Au/PolyA/MCH electrode and incubated in the dark for 1 h to get the GCE/Au/PolyA/MCH/HPV16/H1/H2 electrode. The purpose of this particular assembly was to allow the hybridization between the H1/H2 duplex obtained by the Cu²⁺-dependent DNAzyme-driven CHA and the PolyA probe, thereby modifying the H1/H2 duplex onto the electrode surface. The duration of 1 h in this particular assembly was selected according to the work reported by Cui et al.³² and the volume of 15 μL was determined based on previous work from our group.²⁴ Subsequently, the electrode was immersed in CuSO₄ solution (10 mM, 100 μL) and sodium ascorbate (100 mM, 10 μL) was added for the in situ synthesis of CuNCs. After reaction for 20 min, the obtained electrode was thoroughly rinsed with ultrapure water to remove the remaining species and labeled as the GCE/Au/PolyA/MCH//HPV16/H1/H2/CuNCs electrode. The immersion time (20 min) and the washing step were based on the work reported by Xie et al.³³ Finally, the impedance value of the electrode was measured using EIS.

Results and Discussion

Characterization of CuNCs and the developed electrochemical sensor

The morphology of CuNCs was characterized by transmission electron microscopy (TEM) at a magnification of 1200000×, and the result is shown in Figure 2. We employed the ImageJ 1.50b (National Institute of Health, Bethesda, MD, USA) to measure the sizes of several CuNCs in the TEM image. The average size of the CuNCs was calculated to be approximately 5 nm. It was also noted that the morphology of the CuNCs exhibited elliptical or circular boundaries, demonstrating the successful synthesis of the CuNCs. Each step of the electrode modification process was characterized by EIS. EIS data were presented in the form of Nyquist plots and fitted using an equivalent electrical circuit (EEC) model. The EEC model includes solution resistance (R_s), electron-transfer resistance (R_et), capacitance (CPE), and Warburg impedance (Z_w). The R_et value is associated with the semicircle at high frequencies in the Nyquist plots, which is the most directive and sensitive parameter in response to changes at the electrode/solution interface. All experimental Nyquist plots obtained in this work were fitted using the online electrochemical analysis tool ImpedAnt.³⁴ The extracted parameters for the EEC model along with their respective error values for the Nyquist plots in Figure 3A, are provided in Table S1 (Supplementary Information (SI) section). As shown in Figure 3A, the GCE/Au electrode exhibited excellent electron-transfer performance, as evidenced by the nearly linear Nyquist curve (R_et = 80 Ω, curve a). After incubation with the PolyA DNA probe, the R_et value increased (R_et = 428 Ω, curve b) due to the covalent binding between the PolyA sequence and AuNPs. The repulsion between the negatively charged phosphate backbone of the PolyA DNA and [Fe(CN)₆]^3-/4- probes hindered the electron-transfer. The further modification with MCH increased the electron transfer resistance by blocking the nonspecific sites (R_et = 1481 Ω, curve c). In the presence of HPV16 DNA, the H1/H2 duplex was formed through the Cu²⁺ dependent DNAzyme-driven CHA process. The H1/H2 duplex was introduced to the electrode and hybridized with the PolyA probe, increasing the electron-transfer resistance (R_et = 2043 Ω, curve d). The H1/H2 duplex brought more negatively charged phosphate backbones to the electrode surface, repelling the electrochemical probe [Fe(CN)₆]^3-/4- through electrostatic repulsion. This resulted an increase in electron-transfer resistance. The GCE/Au/PolyA/MCH/HPV16/H1/H2 electrode was then immersed in the CuSO₄ solution and followed by the addition of sodium ascorbate. The H1/H2 duplex modified on the electrode provided favorable binding sites for copper ions and promoted the in situ synthesis of CuNCs, which resulted in a substantial increase in R_et (R_et = 3126 Ω, curve e). The result was consistent with the previously reported work.³⁵ The large electrical resistance could be caused by the disordered aggregates of CuNCs, which hindered electron transport between the probe and the electrode.³⁶ In the absence of HPV16 DNA, the Cu²⁺-dependent DNAzyme driven CHA process could not be triggered, thereby leading to unsuccessful formation of the H1/H2 duplex. When the solution without the H1/H2 duplex was dropped onto the electrode surface, few CuNCs were synthesized on the electrode interface, which resulted in a lower R_et value (R_et = 2149 Ω, curve f) compared with curve e.

Figure 2
TEM image of CuNCs.

Figure 3
(A) Nyquist plots of different modified electrodes for the construction of the electrochemical sensor: (a) GCE/Au; (b) GCE/Au/PolyA; (c) GCE/Au/PolyA/MCH; (d) GCE/Au/PolyA/MCH/HPV16/H1/H2; (e) GCE/Au/PolyA/MCH/HPV16/H1/H2/CuNCs; (f) GCE/Au/PolyA/MCH/H1/H2/CuNCs. The effects of C_Cu-enzyme (B), C_Cu²⁺ (C), and the concentration of H1 and H2 (D) on the S/N ratio.

Optimization of the reaction parameters

To ensure the optimal performance of the electrochemical sensor for HPV16 DNA detection, several key experimental parameters were systematically optimized. These parameters included the concentration of Cu-enzyme, the concentration of Cu²⁺, and the combined concentration of H1 and H2. The effect of these parameters was ultimately reflected in the improvement of the signal/noise (S/N) ratio. The S/N ratio was defined as the difference between the average impedance of the target DNA sample (average_target) and that of the blank control (average_blank) divided by the standard deviation of the blank (SD_blank). This relationship was expressed by equation 1:

(1) $$\mathrm{S}/\mathrm{N}\text{ratio}=({\text{average}}_{\text{target}}-{\text{average}}_{\text{blank}})/{\mathrm{SD}}_{\text{blank}}$$

First, the concentration of Cu-enzyme was optimized by setting Cu-enzyme concentration (C_Cu-enzyme) gradients at 0.5, 1, 1.5, 2 and 2.5 μM. As shown in Figure 3B, the S/N ratio progressively increased with rising the C_{Cu enzyme} from 0.5 to 2 μM. The reason is that the enzymatic activity and cleavage efficiency of the DNAzyme increased with increasing C_Cu-enzyme, consequently promoting the CHA amplification process and CuNCs synthesis. When C_{Cu enzyme} was 2.5 μM, the S/N ratio decreased. Therefore, 2 μM was selected as the optimal of C_Cu-enzyme for the subsequent experiment. In addition, the concentration of Cu²⁺ (C_Cu²⁺) was also a critical factor which influenced the cleavage efficiency. As shown in Figure 3C, the S/N ratio progressively increased and reached the maximum value at 10 mM with increasing C_Cu²⁺. However, when C_Cu²⁺ was more than 15 mM, a decline in the S/N ratio was observed. Cu²⁺ acted as the cofactor that activated the DNAzyme to cleave the substrate strand at the specific site. When the Cu²⁺ concentration was within the appropriate range, increasing the Cu²⁺ concentration promoted more sufficient cleavage reaction, which increased the amount of S1 strand released and thus improved the S/N ratio. However, when the Cu²⁺ concentration exceeded the optimal range, excess Cu²⁺ would non-specifically interact with the phosphate backbone of the DNAzyme and the substrate strand through electrostatic interaction. The secondary structure of the DNAzyme was thus changed and the binding affinity between the DNAzyme and the substrate was reduced, ultimately inhibiting the cleavage activity of the DNAzyme. The reduced cleavage efficiency could lead to a decrease in the amount of released S1 strand, resulting in a decrease in the final S/N ratio. Therefore, the optimal C_Cu²⁺ was chosen to be 10 mM. The concentrations of H1 and H2 in centrifuge tube 2 were optimized. As shown in Figure 3D, the S/N ratio initially increased and then decreased with increasing concentration of H1 and H2, with the maximum value obtained at 2 μM. The reason may be attributed to the fact that the insufficient H1/H2 could limit the CHA reaction efficiency and result in inadequate formation of the H1/H2 duplex, while excessive H1/H2 could increase the incidence of adverse reactions, thereby compromising the analytical performance. Consequently, the optimal concentration for H1 and H2 was selected to be 2 μM.

Analytical performance of the developed electrochemical sensor for the detection of HPV16 DNA

The analytical performance of the developed electrochemical sensor for HPV16 DNA detection was evaluated using EIS, and the detection concentration of the target ranged from 1 pM to 10 μM. The EEC model for the Nyquist plots in Figure 4A was the same as that of Figure 3A and the extracted parameters, along with their respective error values, are provided in Table S2 (SI section). As shown in Figure 4A, the impedance values gradually increased with raising the concentration of HPV16 DNA. Figure 4B presented a good linear relationship between the logarithm of the HPV16 DNA concentration and the impedance. The linear regression equation was R_et = 144.62 logC + 3183.20, with a correlation coefficient (R²) of 0.9926. The limit of detection (LOD) of the developed sensor was calculated to be 6.83 pM based on equation 2:

Figure 4
(A) Nyquist plots of the developed sensor in the presence of different concentrations of HPV16 DNA, and (B) calibration curve of impedance versus the logarithm of HPV16 DNA concentration.

(2) $$\text{LOD}=3\sigma /S$$

where σ denotes the standard deviation of the blank sample set and S denotes the slope of the fitted line in Figure 4B.³⁷

Moreover, in this section, three independent measurements were performed for each concentration of HPV16 DNA using three different electrodes. The relative standard deviations (RSD) for HPV16 DNA at concentrations of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM were calculated to be 4.6, 5.2, 3.7, 3.9, 3.2, 0.9, 2.4 and 6.3%, respectively. These results indicated that the developed sensor exhibited acceptable reproducibility. The analytical performances of various detection techniques for HPV16 DNA detection, including colorimetric, fluorescence, and electrochemical approaches were compared, and the results are present in Table 1. It is noteworthy that linear range obtained in this work was the widest, and the LOD was also the lowest. The best analytical performance of the developed sensor could be attributed to the efficient target recognition and the signal amplification achieved by the DNAzyme-driven CHA process and the CuNCs.

Table 1
Comparison of the analytical performance of various sensors for HPV16 DNA detection

Technique	Target	LOD	Linear range	Reference
Colorimetric	HPV16	3.5 nM	0.78 nM-15.6 nM	38
Fluorescence	HPV16	7.64 pM	-	39
Fluorescence	HPV16	0.44 nM	0.05 nM-10 nM	40
Fluorescence	HPV16	2 nM	0.1 nM-100 nM	41
Electrochemistry	HPV16	2.39 nM	5 nM-20 nM	42
Electrochemistry	HPV16	1.49 nM	2 nM-10 nM	43
Electrochemistry	HPV16	4 nM	0.02 μM-12 μM	44
Electrochemistry	HPV16	6.83 pM	1 pM-10 μM	this work

Selectivity

Selectivity is one of the key criteria for evaluating sensor performance, referring to the ability to distinguish the analyte from other interferents in a real sample. To validate the selectivity of the developed sensor for HPV16 DNA detection, four common HPV genotypes including HPV18, HPV31, HPV51 and HPV58, were selected as potential interferents for the selectivity experiment. The concentration of HPV16 DNA was 1 μM, while the concentrations of all other interferents were 100 μM. As shown in Figure 5, the sensor produced a small electrochemical impedance toward the interferents and the blank sample, while the HPV16 DNA sample gave a large impedance value. The possible reason could be attributed to the specific recognition effect of the biological identification element on the target analyte, effectively avoiding the interference of other coexisting substances in the detection system. These results demonstrated that the developed sensor exhibited excellent selectivity. However, at concentrations close to the actual levels of HPV16 DNA, the selectivity of the developed sensor was not satisfactory due to the increased probability of false-positives results. We intend to further investigate this issue in future research.

Figure 5
Selectivity of the developed sensor.

Recovery test

To evaluate the practical application capability of the developed sensor, a recovery experiment was performed using bovine serum as the real sample. Different concentrations (1, 10, 100 and 1000 nM) of HPV16 DNA were spiked into 20-fold diluted bovine serum for testing. The results are shown in Table 2, indicating that the recovery rates for HPV16 DNA detection in bovine serum ranged from 89.1 to 95.6% and the RSD ranged from 1.9 to 4.4%. On the basis of the above recovery test results, the developed sensor could be combined with a portable electrochemical workstation to construct a rapid on-site detection system. It is expected to provide a new alternative solution for the early clinical screening of HPV-related diseases.

Table 2
Recovery assays of HPV16 DNA in 20-fold diluted bovine serum (n = 3)

Sample No.	Added / nM	Found / nM	RSD / %	Recovery / %
1	1	0.9	2.0	90.0
2	10	8.9	1.9	89.1
3	100	91.5	4.2	91.5
4	1000	956.1	4.4	95.6

Conclusions

This work integrated three strategies, including the Cu²⁺-dependent DNAzyme catalytic reaction, CHA, and PolyA immobilization, to construct an electrochemical sensor with the cascade signal amplification for the sensitive detection of HPV16 DNA. The efficient target recognition and signal amplification were achieved by the DNAzyme-driven CHA process, while CuNCs, which were in situ synthesized on the electrode, were responsible for the electrochemical signal. The developed sensor exhibited a wide linear range from 1 pM to 10 μM, with a low limit of detection of 6.83 pM, and demonstrated superior discrimination capability against the interfering substances. The recovery rates ranged from 89.1 to 95.6%, showing good practical applicability in real samples. Furthermore, by redesigning the sequences of the DNA strands, the developed sensing platform offeres promising potential for the detection of other nucleic acid.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

Supplementary PDF

Supplementary PDF

Acknowledgments

This research was funded by the Research Foundation for PhD of Xiangtan University (grant No. 18QDZ03).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon reasonable request.

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*

e-mail: [e-mail]

Editor handled this article:

Cristiane Luísa Jost (Associate)

Publication Dates

• Publication in this collection
  21 Sept 2026
• Date of issue
  2026

History

• Received
  22 Apr 2026
• Published
  25 Aug 2026