DNA Probes Research Articles

Ratiometric electrochemical biosensor based on hybridization chain reaction signal amplification for sensitive microRNA-155 detection.

In this work, a sensitive ratiometric electrochemical biosensor for microRNA-155 (miRNA-155) detection is reported based on a hybridization chain reaction amplifying the electrochemical signal. The biosensor was fabricated using Au NPs as a modified material to assemble capture DNA labeled with ferrocene (Fc) molecules, and a DNA probe labeled with methylene blue (MB) was employed for the signal probe. In the presence of target miRNA-155, it can be dual hybridized with capture and signal probe, especially with signal probe to continuously produce long concatemers containing lots of MB molecules. The electrochemical signal of Fc was used for the internal signal, and the signal from MB was used as an indicator signal. As the concentration of miRNA-155 was altered, the internal reference signal of Fc remained constant, and only the indicator signal changed in a sensitive way. The change in the ratio (IMB/IFc) between the indicator signal of MB and internal reference signal of Fc can be used to monitor the concentration of miRNA-155. Under optimal conditions, the prepared ratiometric biosensor could detect miRNA-155 within a wide linear range from 100 fM to 100 nM with low detection limit of 33 fM (at S/N = 3). Moreover, the biosensor was evaluated with human serum samples, and satisfactory recoveries were obtained, indicating that the ratiometric biosensor can be applied to clinical sample analysis.

Label-free and ultrasensitive detection of environmental lead ions based on spatially localized DNA nanomachines driven by hyperbranched hybridization chain reaction

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb2+ was developed by integrating Pb2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb2+-sensing platform was also demonstrated to be applicable for Pb2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental monitoring.

Superparamagnetic photonic crystals with DNA probes for rapid visual detection of mercury

A novel superparamagnetic photonic crystal DNA probe (Fe3O4@SiO2@amino@DNA SPC) was developed to enable rapid visual detection of Hg2+. This unique photonic crystal (PC) was synthesized by combining superparamagnetic nanospheres with DNA probes. The DNA probe, rich in thymine (T), detects mercury ions through base mismatch, resulting in the formation of T–Hg2+–T loop hairpin structures. With the binding of Hg2+ to the probe attached to superparamagnetic nanospheres, the PC structure assembled by these nanospheres, formed by the magnetic field, was changed. This change enhanced the reflection intensity; it could be quantified using a fiber optic spectrometer and was visible to the naked eye. The Fe3O4@SiO2@amino@DNA SPC, specific to Hg2+, exhibited a reflection peak at 679 nm, which intensified with increasing Hg2+ concentration. The reflection intensity increased by 132.58 a.u., and the PC color shifted from red to yellow as the Hg2+ concentration increased from 0.1 μg/L to 1 mg/L.

DSN signal amplification strategy based nanochannels biosensor for the detection of miRNAs

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules.

Enhancing public health safety: Development and application of a MoS2@CNT-Chit electrochemical DNA biosensor for rapid and accurate detection of S. Typhi

This study introduces an advanced electrochemical biosensor that utilizes MoS2@CNT as an electrode material combined with a specific DNA probe to detect Salmonella Typhi rapidly and accurately. The sensor offers a broad detection range from 1.0 × 10−6 to 1.0 × 10−18 molL−1 and boasts an exceptionally low limit of detection (LOD) of 1.0 × 10−20 molL−1 for the target bacterium. It demonstrates a detection range from 1.0 × 104 to 1.0 × 1011 CFUml−1 in real samples, with a corresponding LOD of 1.0 × 104 CFUml−1. Rigorous testing against base mismatches and various bacterial strains confirms its specificity, ensuring reliable performance. Validated in real samples, the biosensor can accurately identify Salmonella Typhi in water and milk, achieving recoveries ranging from 92.95 % to 99.58 %. The exceptional performance of the biosensor is attributed to the MoS2@CNT electrode material and the specific DNA recognition probe, which enhance electron transfer and reduce steric impedance. These improvements contribute to the sensor's enhanced sensitivity and specificity, making it a significant advancement in public health safety by providing a rapid and accurate tool for detecting Salmonella Typhi in food samples.

Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 UmL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

Metal ion-complexed DNA probe coupled with CRISPR/Cas12a amplification and AuNPs for sensitive colorimetric assay of metallothionein in fish

The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg2+ and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion-induced water bio-pollution. The target MT molecules bind Hg2+ in the Hg2+- complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence-containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.

Endogenous mRNA-Driven "One-To-More" Signal Amplification of DNA Probe for Intracellular miR155 Sensing.

The detection of specific intracellular microRNAs could be potentially helpful in understanding the underlying mechanisms of cancer metastasis and invasion. MiRNAs are usually present in lower expression levels, especially in early stage of cancer. Here, we proposed a "one-to-more" amplification strategy for miRNA imaging, by virtue of DNA strand displacements with dual-amplification. This approach involves leveraging high-abundance endogenous mRNA as fuel strand to drive cascade reactions between DNA strands for amplification, enabling the monitoring of low-abundance intracellular microRNA155. Notably, in comparison to the traditional "one-to-one" signal triggering mode, our "one-to-more" amplification strategy led to a remarkable 11.8-fold increase in fluorescence signal. Our approach not only demonstrates a high sensitivity and specificity in detecting miR155, but also allows for discrimination of miR155 expression levels in different cell lines. With the advantages of intracellular signal amplification and reduced background signal, this approach holds substantial potential in the early diagnosis of cancer.

Detection of MicroRNA-155 based on lambda exonuclease selective digestion and CRISPR/cas12a-assisted amplification

In numerous malignancies, miRNA-155 is overexpressed and has oncogenic activity because it is one of the most efficient microRNAs for inhibiting apoptosis in human cancer cells. As a result, the highest sensitive detection of the miRNA-155 gene is a technological instrument that can enable early cancer screening. In this study, a miRNA-155 biosensor was created to create a hairpin probe that can bind to the miRNA-155 gene using lambda nucleic acid exonuclease, which can cut the 5′ phosphorylated double strand, and by the DNA probe is recognized by the Cas12a enzyme, which then activates Cas12a to catalyze trans-cutting produces strong fluorescence. Research finding, the target concentration's logarithm and corresponding fluorescence intensity have a strong linear connection, and the limit of detection (LOD) of the sensing system was determined to be 8.3 pM. In addition, the biosensor displayed exceptional specificity, low false-positive signal, and high sensitivity in detecting the miRNA-155 gene in serum samples. This study's creation of a biosensor that has high sensitivity, good selectivity, and is simple to operate provides promising opportunities for research into biosensor design and early cancer detection.

Electrochemical DNA biosensor for HPV-16 detection based on novel carbon quantum dots/APTES composite nanofilm

Detection of high-risk human papillomavirus (HR-HPV) is critical for early warning and accurate screening of cervical disease in women. Electrochemical biosensors offer a promising analytical strategy that can circumvent the structural complexity of traditional optical components found in current bioassays. In this work, an innovative electrochemical biosensor for HPV-16 detection was developed, utilizing advanced nanomaterial technology and optimized DNA probe grafting processes. The self-assembled composite nanofilm was optimized by conducting an orthogonal analysis. The nanofilm exploited the unique advantages and outstanding performance of (3-aminopropyl)triethoxysilane (APTES) and carbon quantum dots (CQDs), which act as a “molecular bridge” between the biosensing interface and the electrode substrate. The electroactive molecule [Ru(NH3)6]3+ facilitated the highly sensitive signaling of the target DNA by interacting with the double-stranded DNA. Under optimal conditions, the developed electrochemical DNA biosensor exhibited high sensitivity in detecting HPV-16, with a limit of detection (LOD) of 0.73 fM, which showed exceptional potential to be used in widespread point-of-care (PoC) screening programs, opening up new opportunities for various applications.

Dual-Stimuli Regulation of DNAzyme Cleavage Reaction by Coordination-Driven Nanoprobes for Cancer Cell Imaging.

Endowing current artificial chemical reactions (ACRs) with high specificity and intricate activation capabilities is crucial for expanding their applications in accurate bioimaging within living cells. However, most of the reported ACR-based evaluations relied on either single biomarker stimuli or dual activators without obvious biological relevance, still limiting their accuracy and fidelity. Herein, taking the metal-ion-dependent DNAzyme cleavage reaction as a model ACR, two regulators, glutathione (GSH) and telomerase (TE) activated DNAzyme cleavage reactions, were exploited for precise discrimination of cancerous cells from normal cells. DNA probe was self-assembled into the ZIF-90 nanoparticle framework to construct coordination-driven nanoprobes. This approach enhances the stability and specificity of tumor imaging by utilizing biomarkers associated with rapid tumor proliferation and those commonly overexpressed in tumors. In conclusion, the research not only paves the way for new perspectives in cell biology and pathology studies but also lays a solid foundation for the advancement of biomedical imaging and disease diagnostic technologies.

A New SPQC Biosensor for the Detection of a New Target of Escherichia/Shigella Genera Based on a Novel Method of Synthesizing Long-Range DNA.

The rapid and sensitive detection of Escherichia/Shigella genera is crucial for human disease and health. This study introduces a novel series of piezoelectric quartz crystal (SPQC) sensors for detecting Escherichia/Shigella genera. In this innovative biosensor, we propose a new target and novel method for synthesizing long-range DNA. The method relies on the amplification of two DNA probes, referred to as H and P amplification (HPA), resulting in the products of long-range DNA named Sn. The new target was screened from the 16S rRNA gene and utilized as a biomarker. The SPQC sensor operates as follows: the Capture probe is modified on the electrodes. In the presence of a Displace probe and target, the Capture can form a complex with the Displace probe. The resulting complex hybridizes with Sn, bridging the gap between the electrodes. Finally, silver wires are deposited between the electrodes using Sn as a template. This process results in a sensitive response from the SPQC. The detection limit of the SPQC sensor is 1 CFU/mL, and the detection time is within 2 h. This sensor would be of great benefit for food safety monitoring and clinical diagnosis.

Label-free detection of platelet-derived growth factor-BB via liquid crystal/aqueous interface functionalized using a double-stranded DNA probe

A liquid crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with cationic surfactant octyltrimethylammonium bromide (OTAB) and adsorbed with a double-stranded deoxyribonucleic acid (dsDNA) probe at the LC/aqueous interface (TEMOTAB/DNA) was utilized for the highly specific detection of platelet-derived growth factor (PDGF-BB). Upon adsorption of the OTAB-coated 4-cyano-4′-pentylbiphenyl (5CB) on the TEM grid (TEMOTAB) and subsequent adsorption of the dsDNA probe, a homeotropic orientation was observed. Subsequently, the TEMOTAB/DNA biosensor was exposed to PDGF-BB, which induced a homeotropic-to-planar configurational change in 5CB, as observed using polarized optical microscopy with crossed polarizers. The optimized adsorption density of the dsDNA probe facilitated the detection of 2.63 pg mL−1 PDGF-BB in the detection range of 5–200 pg mL−1. Remarkably, this LC-based biosensor discriminated PDGF-BB from bovine serum albumin, immunoglobulin G, hemoglobin, carcinoembryonic antigen, epidermal growth factor, PDGF-AA, and PDGF-AB. The application potential of this sensor was further demonstrated by its effective performance in detecting PDGF-BB in both human serum and synthetic urine samples. Because of its high specificity, sensitivity, and label-free detection capabilities, this biosensor has potential applications in cancer detection.

Re-Examination of PGT-A Detected Genetic Pathology in Compartments of Human Blastocysts: A Series of 23 Cases.

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments-the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed.

Rapid and Label-Free Detection of Influenza A Virus With a SAW Biosensor Based on DNA Probes

Rapid and Label-Free Detection of Influenza A Virus With a SAW Biosensor Based on DNA Probes

Optimization of Fluorescence In Situ Hybridization Protocols in the Era of Precision Medicine.

Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy

The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values < 0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.

Development of label-free electrochemical OMP-DNA probe biosensor as a highly sensitive system to detect of citrus huanglongbing

The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Construction of Multiply Guaranteed DNA Sensors for Biological Sensing and Bioimaging Applications.

Nucleic acids exhibit exceptional functionalities for both molecular recognition and catalysis, along with the capability of predictable assembly through strand displacement reactions. The inherent programmability and addressability of DNA probes enable their precise, on-demand assembly and accurate execution of hybridization, significantly enhancing target detection capabilities. Decades of research in DNA nanotechnology have led to advances in the structural design of functional DNA probes, resulting in increasingly sensitive and robust DNA sensors. Moreover, increasing attention has been devoted to enhancing the accuracy and sensitivity of DNA-based biosensors by integrating multiple sensing procedures. In this review, we summarize various strategies aimed at enhancing the accuracy of DNA sensors. These strategies involve multiple guarantee procedures, utilizing dual signal output mechanisms, and implementing sequential regulation methods. Our goal is to provide new insights into the development of more accurate DNA sensors, ultimately facilitating their widespread application in clinical diagnostics and assessment.

Zeolite-iron oxide integrated interdigitated electrode sensor for diagnosing cervical cancer

Cervical cancer is caused by changes in the cervix that lead to precancerous cells and eventually progress to cancer. Human papillomavirus (HPV) infections are the primary cause of cervical cancer. Early detection of HPV is crucial in preventing cervical cancer, and regular screening for HPV infection can identify cell changes before they develop into cancer. While Pap smear tests are reliable for cervical cancer screening, they are critical, expensive, and labor-intensive. Therefore, researchers are focusing on identifying blood-based biomarkers using biosensors for cervical cancer screening. HPV strains 16, 45, and 18 are common culprits in cervical cancer. This study aimed to develop an HPV-16 DNA biosensor on a zeolite-iron oxide (zeolite-IO) modified interdigitated electrode (IDE) sensor. The DNA probe was immobilized on the IDE through amine-modified zeolite-IO, enhancing the hybridization of the target and DNA probe. The detection limit of the DNA-DNA duplex was found to be 7.5 pM with an R2 value of 0.9868. Additionally, control experiments with single and triple mismatched sequences showed no increase in current responses, and the identification of target DNA in a serum-spiked sample indicated specific and selective target identification.