Label-free Detection Method Research Articles

A novel approach ofdifferentiation of adenoma and carcinoma in lung cancer based on biogenic in situ synthesis of gold nanostructures on various oligonucleotide motifs.

Aunique approach is introducedfor constructing gold nanocrystals (AuNCs) with RNA motif-directed morphologies in a sequence-independent manner and itsapplications in theclinical areaare described. By using this method, a label-free LSPR-based detection method for the SOX2OT transcript, long non-coding RNAs (lncRNAs), which is a prognostic indicator of poor survival in lung cancer patientsis presented. For the first time, we examined how the structural changes of RNA after the heteroduplex formation with a specific DNA probe can change the morphology and LSPR band of AuNCs. Using this method, is was possible to differentiate lung squamous cell carcinoma from adenocarcinoma samples without a need for a prior amplification of the target lncRNA. The approach of using specific DNA probe enables the in situ synthesis of nanocrystals in a different way and expands this method for future translational medicine, particularly detection of specific RNA.

A Lab Prototype for Rapid Electrochemical Detection of Escherichia coli in Water Using Modified Screen-Printed Electrodes.

Recognizing the need for a hand-held device capable of quantitatively measuring the concentration of bacteria in water, this paper describes a label-free method for rapid detection of Escherichia coli (E. coli) in water via H2O2 decomposition using screen-printed electrodes (SPE) modified with nanostructured metal oxide layers. The study encompasses sensor preparation, bacteria culture, synthesis and characterization of nanostructures, and development of a readout circuitry for lab prototyping. During sensing measurements, the bacteria are first made to interact with H2O2 and subsequently, the H2O2 solution is exposed on the sensing surface. The electrochemical sensors are fabricated by modifying the working electrode of SPE with nanostructured metal oxide layers of MnO2 and TiO2 as these play a crucial role in the detection of E. coli in water. The sensing experiments of MnO2-modified SPE show a significant response to bacteria with a sensitivity of 0.82 mV.mL/log CFU and a limit of detection (LOD) of 1.8 CFU/mL, while the TiO2-modified SPE exhibits a linear response over a wide range of bacterial concentrations with a sensitivity of 1.12 mV·mL/log CFU and a limit of detection of 2.23 CFU/mL. Both sensors demonstrate a rapid response, stability, repeatability, and a recovery time of 70 ms. Additionally, selectivity with respect to other bacteria, wastewater components such as glucose, ammonium sulfate, and sodium carbonate, and testing with RO, DI, and tap water samples are conducted to evaluate the sensors' performance. A detailed sensing mechanism has been developed to comprehend the results, including chemical and biological reactions, metal oxide interfaces, morphology, and other surface studies of the sensing surface. A prototype comprising a sensor chip, an Arduino board, and other necessary circuit components is tested with various bacterial solutions. This enables its use for on-field rapid detection of bacteria in water using smaller volumes and a portable system.

An enzyme-free and label-free multiplex detection of miRNAs by entropy-driven circuit coupled with capillary electrophoresis

MicroRNAs (miRNAs) are currently recognized as important biomarkers for the early diagnosis and prognostic treatment of cancer. Herein, we developed a simple and label-free method for the multiplex detection of miRNAs, based on entropy-driven circuit (EDC) amplification and non-gel sieving capillary electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform. In this system, three different lengths of fuel chains were designed to catalyze three EDC, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. In the presence of target miRNA, the EDC cycle amplification reaction was triggered, generating numerous stable double-strands products (F-DNA/L-DNA). Since the three miRNAs correspond to three different lengths of F-DNA/L-DNA, they can be easily isolated and detected by NGCE. This strategy has good sensitivity, with detection limits of 68 amol, 292.2 amol, and 394 amol for miRNA-21, miRNA-155, and miRNA-10b, respectively. Additionally, this method has good specificity and can effectively distinguish single-base mismatches of miRNA. The recoveries of the three miRNAs in deproteinized healthy human serum ranged from 91.28 % to 108.4 %, with a relative standard deviation (RSD) of less than 7.9 %. This method was further applied to detect cellular miRNAs in human breast cancer (MCF-7) cell extracts, revealing an up-regulation of miRNA-21, miRNA-155, and miRNA-10b in MCF-7 cells. The successful spiked recovery in human serum and RNA extraction from MCF-7 cells underscores the practicality of this method. Therefore, this strategy has broad application prospects in biomedical research.

Binding-induced activation of CRISPR-Cas12a using PAM-engineered toehold switch and DNA tile architectures for enhanced detection of thrombin

This study presents a novel biosensor that integrates CRISPR-Cas12a technology with DNA tile-based architectures to enhance thrombin detection. Utilizing PAM-engineered toehold switches, the biosensor dynamically activates the CRISPR-Cas12a system upon thrombin recognition, significantly improving both sensitivity and specificity. The label-free electrochemical detection method offers rapid, cost-effective analysis, making it ideal for real-time clinical applications. The biosensor achieved an impressive detection limit of 103 aM, surpassing traditional methods and setting a new standard for thrombin assays. The biosensor's robust performance in complex biological samples, such as diluted human serum, demonstrates its reliability for clinical diagnostics. This is particularly important for the early diagnosis and management of thrombotic and coagulation-related disorders. By combining advanced genetic engineering with traditional electrochemical techniques, the biosensor not only optimizes detection but also expands its potential applications across various biomedical fields. Overall, this biosensor provides a powerful and innovative tool for thrombin detection, offering enhanced diagnostic accuracy, faster patient management, and potential cost savings in healthcare. Its superior performance and adaptability highlight its promise for broader adoption in clinical diagnostics and other biosensing applications.

High-performance electrolyte-gated amorphous InGaZnO field-effect transistor for label-free DNA sensing

Accurate, convenient, label-free, and cost-effective biomolecules detection platforms are currently in high demand. In this study, we showcased the utilization of electrolyte-gated InGaZnO field-effect transistors (IGZO FETs) featuring a large on-off current ratio of over 106 and a low subthreshold slope of 78.5 mV/dec. In the DNA biosensor, the modification of target DNA changed the effective gate voltage of IGZO FETs, enabling an impressive low detection limit of 0.1 pM and a wide linear detection range from 0.1 pM to 1 μM. This label-free detection method also exhibits high selectivity, allowing for the discrimination of single-base mismatch. Furthermore, the reuse of gate electrodes and channel films offers cost-saving benefits and simplifies device fabrication processes. The electrolyte-gated IGZO FET biosensor presented in this study shows great promise for achieving low-cost and highly sensitive detection of various biomolecules.

Label-free fluorescent biosensor based on AuNPs etching releasing signal for miRNA-155 detection

Quantitative microRNA (miRNA) detection is crucial for early breast cancer diagnosis and prognosis. However, quick and stable fluorescence sensing for miRNA identification is still challenging. This work developed a novel label-free detection method based on AuNPs etching for quantitatively detecting miRNA-155. A layer of AuNPs was grown on the surface of mesoporous silica nanoparticles (MSN) loaded with Rhodamine 6G (R6G) using seed-mediated growth, followed by probe attachment. In the presence of miRNA-155, the MSN@R6G@AuNP surface loses the protection of the attached probe, rendering AuNPs susceptible to etching by hydrochloric acid. This results in a significant fluorescent signal being released in the free space. The encapsulation with AuNPs effectively reduces signal leakage, while the rapid etching process shortens detection time. This strategy enables sensitive and fast detection with a detection range of 100 fM to 100 nM, a detection limit of 2.18 fM, and a detection time of 30 min. The recovery rate in normal human serum ranges from 99.02 % to 106.34 %. This work presents a simple biosensing strategy with significant potential for application in tumor diagnosis.

A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

Real-time, ultra-sensitive and label-free detection of OTA based on DNA aptamer functionalized carbon nanotube field-effect transistor

Ochratoxin A (OTA), widely recognized as a mycotoxin contaminant across various food products, poses significant health risks attributed to its nephrohepatotoxic and carcinogenic effects. Consequently, detecting OTA is crucial for ensuring food safety and preventing mycotoxicosis. In our study, we employed a carbon nanotube field-effect transistor (CNT FET) functionalized with DNA aptamers for targeted OTA detection. The sensor achieved a remarkably low detection threshold of 0.2 femtomolars (fM) in phosphate-buffered saline (PBS), enabling real-time detection of OTA over a broad concentration spectrum spanning from 8 fM to 80 pM. This research confirmed the sensor’s superior selectivity by effectively distinguishing OTA from non-target fungal toxins and showcased its rapid analysis capabilities across diverse complex matrices. Utilizing homogenized liquids from ten representative food samples for analysis, the sensor delivered real-time responses within 100 s, accurately identifying varying OTA concentrations down to 80 fM. In conclusion, this biosensor provides a label-free, highly sensitive, and rapid detection method for OTA, introducing novel solutions for effective mycotoxin surveillance in food safety. This biosensor also paves the way for the creation of a multiplex platform for concurrent monitoring of multiple fungal toxins.

Characteristic fingerprint spectrum of α-synuclein mutants on terahertz time-domain spectroscopy

α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is involved in the pathogenesis of Parkinson’s disease. Point mutations and multiplications of α-synuclein (A30P and A53T) are correlated with early-onset Parkinson’s disease characterized by rapid progression and poor prognosis. Currently, the clinical identification of SNCA variants, especially disease-related A30P and A53T mutants, remains challenging and also time consuming. This study aimed to develop a novel label-free detection method for distinguishing the SNCA mutants using transmission terahertz (THz) time-domain spectroscopy. The protein was spin-coated onto the quartz to form a thin film, which was measured using THz time-domain spectroscopy. The spectral characteristics of THz broadband pulse waves of α-synuclein protein variants (SNCA wild type, A30P, and A53T) at different frequencies were analyzed via Fourier transform. The amplitude A intensity (AWT, AA30P, and AA53T) and peak occurrence time in THz time-domain spectroscopy sensitively distinguished the three protein variants. The phase φ difference in THz frequency domain followed the trend of φWT > φA30P > φA53T. There was a significant difference in THz frequency amplitude A′ corresponding to the frequency ranging from 0.4 to 0.66 THz (A′A53T > A′A30P > A′WT). At a frequency of 0.4–0.6 THz, the transmission T of THz waves distinguished three variants (TA53T > TA30P > TWT), whereas there was no difference in the transmission T at 0.66 THz. The SNCA wild-type protein and two mutant variants (A30P and A53T) had distinct characteristic fingerprint spectra on THz time-domain spectroscopy. This novel label-free detection method has great potential for the differential diagnosis of Parkinson’s disease subtypes.

Label-free and selective heparin detection by surface functionalized fiber Fabry-Perot interferometer biosensor

Heparin (Hep) is the most commonly used anticoagulant drug in clinical settings, and precise dosage control is crucial. To enable rapid and accurate quantification of Hep concentration in clinical practice, a label-free and selective fiber-optic Fabry-Perot interferometric (FPI) biosensor detection method based on graphene oxide (GO) was proposed. This method utilizes protamine (PRTM) sulfate as the modified layer and achieves Hep detection through layer-by-layer (LBL) assembly. In this study, we developed a label-free and selective fiber optic FPI biosensor for the detection of Hep using GO. The GO on the surface layer of the fiber optic FPI biosensor was treated with specific functionalization, it enables specific interaction with Hep. The binding of Hep to the functionalized surface induces a change in the luminal light signal, which can be monitored within the cavity for rapid and accurate detection of Hep. The results demonstrate that this biosensor exhibits good sensitivity, with a value of 10.82 nm/mM in the low-concentration control group, and an average limit of detection (LOD) calculated as 31μg/mL in the low-concentration group. This study presents a label-free, selective and efficient method for biomedical science testing that holds great potential for widespread application in clinical medicine.

One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe

In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0–10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

Label-Free, Sensitive, and Versatile Colorimetric Method for Molecule Detection via the G-Quadruplex-Based Signal Quenching Strategy.

Signal amplification strategies have emerged as a prominent tool in the field of improving the detection sensitivity of small extracellular vesicles (sEVs). It is important to highlight that the utilization of signal quenching strategies is not commonly implemented. A detection technique for sEVs was established based on the unwinding of G-quadruplex using Klenow fragment polymerase (KF), which served as an inspiration for this study. This system is characterized by its simplicity and lack of labeling, making it an efficient approach for signal quenching. In the presence of sEVs, the CD63 aptamer in the capture@sMBs complex binds with the CD63 protein on the surface of sEVs to release trigger sequences, which were employed as a primer to mediate the DNA polymerase/endonuclease-assisted signal recycling. The signal recycling process produces numerous single-stranded DNA sequences that can bind to the toehold section of the G-quadruplex. This leads to the rupture of the G-quadruplex structure and the subsequent deactivation of a DNAzyme generated by the G-quadruplex structure and hemin, thereby inhibiting its biological catalytic function. Consequently, the G-quadruplex structure would undergo a transformation to a duplex structure, leading to the emergence of a discernible differential signal that can be noticed in a majority of instances, even without the aid of magnification devices. The decrease in the prominent signal allows for the efficient analysis of target sEVs, which exhibit a notably low detection limit. In addition to the detection of sEVs, the approach has also been utilized for the investigation of miRNA-21. The approach demonstrates a high level of selectivity and robustness in its capacity to differentiate between target miRNA and base-mismatched miRNA as well as other miRNA families. This statement suggests that the assay holds significant promise for use in biochemical research and clinical diagnosis.

Imaging the intracellular refractive index distribution (IRID) for dynamic label-free living colon cancer cells via circularly depolarization decay model (CDDM).

Label-free detection of intracellular substances for living cancer cells remains a significant hurdle in cancer pathogenesis research. Although the sensitivity of light polarization to intracellular substances has been validated, current studies are predominantly focused on tissue lesions, thus label-free detection of substances within individual living cancer cells is still a challenge. The main difficulty is to find specific detection methods along with corresponding characteristic parameters. With refractive index as an endogenous marker of substances, this study proposes a detection method of intracellular refractive index distribution (IRID) for label-free living colon cancer (LoVo) cells. Utilizing the circular depolarization decay model (CDDM) to calculate the degree of circular polarization (DOCP) modulated by the cell allows for the derivation of the IRID on the focal plane. Experiments on LoVo cells demonstrated the refractive index of single cell can be accurately and precisely measured, with precision of 10-3 refractive index units (RIU). Additionally, chromatin content during the interphases (G1, S, G2) of cell cycle was recorded at 56.5%, 64.4%, and 71.5%, respectively. A significantly finer IRID can be obtained compared to the phase measurement method. This method is promising in providing a dynamic label-free intracellular substances detection method in cancer pathogenesis studies.

Quantification of Polystyrene Uptake by Different Cell Lines Using Fluorescence Microscopy and Label-Free Visualization of Intracellular Polystyrene Particles by Raman Microspectroscopic Imaging.

Environmental pollution caused by plastic is a present problem. Polystyrene is a widely used packaging material (e.g., Styrofoam) that can be broken down into microplastics through abrasion. Once the plastic is released into the environment, it is dispersed by wind and atmospheric dust. In this study, we investigated the uptake of polystyrene particles into human cells using A549 cells as a model of the alveolar epithelial barrier, CaCo-2 cells as a model of the intestinal epithelial barrier, and THP-1 cells as a model of immune cells to simulate a possible uptake of microplastics by inhalation, oral uptake, and interaction with the cellular immune system, respectively. The uptake of fluorescence-labeled beads by the different cell types was investigated by confocal laser scanning microscopy in a semi-quantitative, concentration-dependent manner. Additionally, we used Raman spectroscopy as a complementary method for label-free qualitative detection and the visualization of polystyrene within cells. The uptake of polystyrene beads by all investigated cell types was detected, while the uptake behavior of professional phagocytes (THP-1) differed from that of adherent epithelial cells.

EVATOM: an optical, label-free, machine learning assisted embryo health assessment tool

The combination of a good quality embryo and proper maternal health factors promise higher chances of a successful in vitro fertilization (IVF) procedure leading to clinical pregnancy and live birth. Of these two factors, selection of a good embryo is a controllable aspect. The current gold standard in clinical practice is visual assessment of an embryo based on its morphological appearance by trained embryologists. More recently, machine learning has been incorporated into embryo selection “packages”. Here, we report EVATOM: a machine-learning assisted embryo health assessment tool utilizing an optical quantitative phase imaging technique called artificial confocal microscopy (ACM). We present a label-free nucleus detection method with, to the best of our knowledge, novel quantitative embryo health biomarkers. Two viability assessment models are presented for grading embryos into two classes: healthy/intermediate (H/I) or sick (S) class. The models achieve a weighted F1 score of 1.0 and 0.99 respectively on the in-distribution test set of 72 fixed embryos and a weighted F1 score of 0.9 and 0.95 respectively on the out-of-distribution test dataset of 19 time-instances from 8 live embryos.

Directly Self-Assembly of Aligned Ag NWs Films at the Air-Water Interface for the Detection of Pathogens in Artificial Breath Aerosols.

Exhaled aerosols from humans, containing various pathogens, are crucial for early disease diagnosis. However, the traditional pathogen detection methods, such as polymerase chain reaction, are often slow and cumbersome due to complex sampling and procedures. This study introduces a novel, direct, and label-free detection method for pathogens in respiratory aerosols, utilizing a highly aligned silver nanowire (Ag NW) film combined with a filter membrane (Ag NWs@filter) as a surface-enhanced Raman spectroscopy-active substrate. A large-scale, ordered silver nanowire film was developed through a simplified self-assembly process. This process eliminates the need for an organic phase and complex surface modifications of Ag NWs, which are common in other preparation methods. Subsequently, the fabricated Ag NWs@filter demonstrated its capability to continuously capture and efficiently preconcentrate pathogens from aerosols, achieving a remarkable detection limit of 3 × 103 CFU/mL, demonstrated using Escherichia coli (E. coli) as a model pathogen. Moreover, the classification between E. coli and Pseudomonas aeruginosa achieved an overall accuracy of 96.5% by the principal component analysis with linear discriminant analysis models. The success of this sensing strategy illustrates its potential in detecting and identifying a variety of biomarkers present in respiratory aerosols, marking a significant step forward in the field of pathogen detection.

A simple and label-free proximity-catalytic hairpin reaction-based method for sensitive and miRNA analysis

Aberrant expression of microRNAs (miRNAs) has been shown to be linked to several crucial biological processes, including as carcinogenesis, metastasis, and progression. The advancement of innovative miRNA detection technologies can enhance the early detection of malignancies by merging with conventional diagnostic methods, such as ultrasound technology. Herein, we reported a simple, sensitive, and label-free miRNA detection method by integrating the proximity-catalytic hairpin assembly (proximity-CHA) and DNAzyme-assisted signal amplification. Compared with traditional CHA, in which the signal amplification efficiency is greatly limited by the concentration of hairpin probes, the proposed method possesses a greatly improved signal amplification efficiency. The target facilitated the non-enzymatic CHA-driven sequential formation of DNAzyme nanostructures, resulting in the effective DNAzyme-facilitated cleavage of a substrate modified with a fluorophore and quencher, leading to the production of an intensified fluorescence signal. The proximity-CHA-DNAzyme system possesses appealing analytical characteristics, making it highly promising for the analysis of many analytes in clinical research domains.

Label-Free and Real-Time Optical Detection of Affinity Binding of the Antibody on Adherent Live Cells.

Oblique-incidence reflectivity difference (OIRD) is a novel real-time, label-free, and nondestructive optical detection method and exhibits encouraging application in the detection of antibody/DNA microarrays. In this study, for the first time, an OIRD label-free immunoassay was achieved by using adherent live cells as the probe. The cells were cultured on glass cells, and the affinity binding of antibodies targeted on the HLA class I antigen of the cell surface was detected with an OIRD. The results show that an OIRD is able to detect the binding process of anti-human HLA-A, B, and C antibodies on MDA-MB-231 cells and HUVEC cells. Control experiments and complementary fluorescence analysis confirmed the high detection specificity and good quantitative virtue of the OIRD label-free immunoassay. Label-free OIRD imaging analysis of cell microarrays was further demonstrated successfully, and the underlying optical mechanism was revealed by combining the theoretical modeling. This work explores the use of live cells as probes for an OIRD immunoassay, thus expanding the potential applications of the OIRD in the field of pathological analysis, disease diagnosis, and drug screening, among others.

Selection of DNA aptamers for detecting metronidazole and ibuprofen: two common additives in soft drinks.

To enhance the effects of some functional soft drinks, drugs, especially metronidazole (MNZ) and ibuprofen (IBF), are often illegally added. This poses a serious threat to the health of consumers. Therefore, developing simple and rapid detection methods for these additives is crucial. In this study, DNA aptamers of metronidazole and ibuprofen were selected using the library-immobilized method. The best aptamer for metronidazole, named MNZ-1, has a dissociation constant (Kd) value of 4.9 μM and the aptamer for ibuprofen, named IBF-1, shows a Kd of 9.3 μM, as determined by the thioflavin T (ThT) fluorescence assay. The Kd values measured using isothermal titration calorimetry (ITC) were 17.0 μM and 66.7 μM for these two aptamers, respectively. Selectivity experiments indicate that MNZ-1 demonstrates very weak binding to structurally similar drugs, whereas IBF-1 exhibits binding capability to some structurally similar compounds comparable to ibuprofen, enabling the simultaneous detection of these types of drugs. Neither MNZ-1 nor IBF-1 binds to other common drugs. Using ThT, a label-free fluorescent detection method was developed for metronidazole and ibuprofen in soft drinks, showing limits of detection (LODs) of 0.6 μM and 4.7 μM, respectively. Owing to their small size and well-defined secondary structures, these aptamers are expected to be utilized in analytical applications for food and environmental monitoring.

Label-free SERS method with size-matched selectivity for analytes of varying sizes

Label-free Surface-enhanced Raman spectroscopy (SERS) methods have great potential for detecting analytes of various sizes and dimensions with high sensitivity and selectivity. To mitigate the impact of interfering substances, SERS-active substrates with size-matched selectivity were designed by coating Au nanoparticles (Au NPs) on ZrO2 multilayer nanofibers (Au NPs/fZrO2) and referencing with Au NPs embedded on ZrO2 nanobowl (Au NPs/pZrO2). The detection efficiency study covered four small pesticide molecules, two live SARS-CoV-2 virus variants (Alpha and Delta), and three interfering substances. The results demonstrate that the fibrous structure of Au NPs/fZrO2 could effectively wet the sample and detect larger target molecules, such as live SARS-CoV-2 virus, thus improving efficiency by reducing unwanted molecules in the SERS signal. This substrate also showed high discrimination between Alpha and Delta variants. Au NPs/fZrO2 demonstrated similar trace detection capabilities to Au NPs/pZrO2 for pesticide molecules and virus variants; however, it particularly exhibited stronger peak intensities and more unique SERS peaks assigned to the variants. Thus, differences in substrate morphology affect the generation of hotspots and the distance between analyte and hotspots. These findings hold promise for the development of SERS-based label-free analytical method for trace detection of a variety of virus particles.