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Colorectal cancer (CRC) remains a significant contributor to the global mortality rate, and a single biomarker cannot meet the specificity required for CRC screening. To this end, we developed a multiplexed, pump-free surface-enhanced Raman scattering (SERS) microfluidic chip (LoC-SERS) using a one-step recognition release mechanism; the aptamer-functionalized novel Au nanocrown array (AuNCA) was used as the detection element embedded in the detection zone of the platform for rapid and specific detection of protein markers in multiple samples simultaneously. Here, the corresponding aptamer specifically captured the protein marker, causing the complementary strand of the aptamer carrying the Raman signal molecule to be shed, reducing the SERS signal. Based on this platform, sensitive and specific detection of the target can be accomplished within 15 min with detection limits of 0.031 pg/mL (hnRNP A1) and 0.057 pg/mL (S100P). Meanwhile, the platform was consistent with ELISA results when used to test clinical. By substituting different aptamers, this platform can provide a new solution for the rapid and sensitive detection of protein markers, which has promising applications in future disease detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged as a novel pathogen in 2019. The virus is responsible for a severe acute respiratory syndrome outbreak, affecting the respiratory system of infected individuals. COVID-19 is a super amplifier of basic diseases, and the disease with basic diseases is often more serious. Controlling the spread of the COVID-19 pandemic relies heavily on the timely and accurate detection of the virus. To resolve the problem, a polyaniline functionalized NiFeP nanosheet array-based electrochemical immunosensor using Au/Cu 2 O nanocubes as a signal amplifier is fabricated for the detection of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP). Polyaniline (PANI) functionalized NiFeP nanosheet arrays are synthesized as an ideal sensing platform for the first time. PANI is coated on the surface of NiFeP by electropolymerization to enhance biocompatibility, beneficial for the efficient loading of the capture antibody (Ab 1 ). Significantly, Au/Cu 2 O nanocubes possess excellent peroxidase-like activity and exhibit outstanding catalytic activity for the reduction of H 2 O 2 . Therefore, Au/Cu 2 O nanocubes combine with a labeled antibody (Ab 2 ) through the Au-N bond to form labeled probes, which can effectively amplify current signals. Under optimal conditions, the immunosensor for the detection of SARS-CoV-2 NP shows a wide linear range of 10 fg mL -1 -20 ng mL -1 and a low detection limit of 1.12 fg mL -1 (S/N = 3). It also exhibits desirable selectivity, repeatability, and stability. Meanwhile, the excellent analytical performance in human serum samples confirms the practicality of the PANI functionalized NiFeP nanosheet array-based immunosensor. The electrochemical immunosensor based on the Au/Cu 2 O nanocubes as a signal amplifier demonstrates great potential for application in the personalized point-of-care (POC) clinical diagnosis.

Purpose: Circulating tumor DNA (ctDNA) is more representative and accurate than biopsy and is also conducive to dynamic monitoring, facilitating accurate diagnosis and prognosis of glioma. Therefore, the present study aimed to establish and validate a novel amplified method for the detection of IDH1 R132H and BRAF V600E, which were associated with the genetic diagnosis of glioma., Patients and Methods: A dual-signal amplification method based on magnetic aggregation and catalytic hairpin assembly (CHA) was constructed for the simultaneous detection of ctDNAs. When target ctDNAs are present, the CHA reaction is initiated and leads to the assembly of Au-Ag nanoshuttles (Au-Ag NSs) onto magnetic beads (MBs). Further enrichment of MBs under an external magnetic field facilitated the dual-signal amplification of SERS., Results: The limit of detection (LOD) for IDH1 R132H and BRAF V600E in serum was as low as 6.01 aM and 5.48 aM. The reproducibility and selectivity of the proposed SERS analysis platform was satisfactory. Finally, the platform was applied to quantify IDH1 R132H and BRAF V600E in the serum of subcutaneous-tumor‑bearing nude mice, and the results obtained by SERS were consistent with those from quantitative real-time polymerase chain reaction (qRT-PCR)., Conclusion: The present study showed that the dual-signal amplification method is a simple and ultrasensitive strategy for gliomas-associated ctDNAs detection, which is crucial for early diagnosis and dynamic monitoring., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Wang et al.)

Multiplexed detection of Parkinson's disease (PD) biomarkers is of great significance for early diagnosis and personalized treatment. In this study, we fabricated a robust surface-enhanced Raman scattering-enabled lab-on-a-chip (LoC-SERS) platform for simultaneous quantification of α-synuclein, phosphorylated tau protein 181, osteopontin, and osteocalcin. Herein, the antibody-DNA conjugate was designed to introduce the catalytic hairpin self-assembly (CHA) amplification into the protein detection. Au nano-stars (AuNSs) modified with Raman reporter molecules and hairpin-structure DNA 1 were applied as the SERS nanotags. Au-coated silicon nanocone array (Au/SiNCA) fabricated based on the maskless plasma etching-prepared high-density Si nanocone array (SiNCA) and surface ion sputtering was used as the capture substrate after the modification of hairpin-structure DNA 2. Benefitting from the antibody-DNA conjugate-induced CHA amplification, numerous AuNSs can be connected to the Au/SiNCA surface, which significantly amplify the plasmonic coupling effect for ultrasensitive SERS detection, and the limit of detection was less than the pg/mL level. The application of highly uniform Au/SiNCA and antibody-DNA conjugate endows the LoC-SERS platform excellent analytical performance, including superior reproducibility, satisfactory universality, and high sensitivity. In addition, a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model was established, and satisfactory results were obtained in real sample analysis with the LoC-SERS platform, which may be enlightening for exploiting protein biomarkers in PD monitoring.

In this work, a microfluidic chip using Au@SiO 2 array-based highly active SERS substrates was developed for quantitative detection of squamous cell carcinoma antigen (SCCA) and carcinoembryonic antigen (CEA) associated with cervical cancer. The chip consisted of six functional units with pump-free design, enabling parallel detection of multiple samples in an automatic manner without external pumps and improving the portability. Ag nanocubes (AgNCs) were labeled with Raman reporters and coupled with antibodies (labeling) to prepare SERS tags, while the Au nanoparticle-modified SiO 2 microsphere (Au@SiO 2 ) array was conjugated with antibodies (coating) to generate the highly SERS-active capturing substrate. In the presence of target biomarkers, they were captured by SERS tags and capturing substrate, resulting in the formation of "sandwich" structures which were trapped in the detection chamber. As the immune reaction proceeded, a large number of "hot spots" were generated by the proximity of the Au@SiO 2 array substrate and AgNCs, greatly amplifying SERS signals. With this chip, the limits of detection of the SCCA and CEA levels in human serum were estimated to be as low as 0.45 pg mL -1 and 0.36 pg mL -1 , respectively. Furthermore, the good selectivity and reproducibility of this chip were confirmed. Finally, clinical serum samples were analyzed by this chip, and the outcomes were consistent with those of enzyme-linked immunosorbent assay (ELISA). Thus, the proposed microfluidic chip can be potentially applied for the clinical diagnosis of cervical cancer., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

MicroRNA (miRNA), as one of the ideal target biomarker analytes, plays an essential role in biological processes; thus, the development of rapidly sensitive detection methods is imperative. Herein, we proposed a pump-free surface-enhanced Raman scatting (SERS) microfluidic chip for the rapid and ultrasensitive detection of miR-106b and miR-196b, laryngeal squamous cell carcinoma (LSCC)-related miRNAs. Ag-Au core-shell nanorods (Ag-AuNRs) were applied to prepare SERS tags by modifying Raman reporters and hairpin DNAs. The capture probes were synthesized by labeling hairpin DNAs onto the magnetic beads (MBs) surface. In the presence of targets, the catalytic hairpin assembly (CHA) reactions between SERS tags and capture probes could be triggered, causing the aggregation of Ag-AuNRs. The tiny magnets installed under the rectangular chamber could magnetically gather the CHA products, leading to the further aggregation of Ag-AuNRs. Thus, this strategy could achieve the double aggregation of Ag-AuNRs, resulting in the significant amplification of the SERS signal. The proposed strategy achieved simultaneous and sensitive detection of miR-106b and miR-196b, with limits of detection low to aM level. The whole detection process could be completed within 5 min. Moreover, this microfluidic chip exhibited excellent reproducibility, stability, and specificity. The high accuracy of this SERS microfluidic chip was proved by practical analysis in LSCC patients' serum. The results demonstrated that SERS could be a promising alternative clinical diagnosis tool and exhibited potential application for the dynamic monitoring of cancer staging., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Accurate quantification of multiple miRNAs biomarkers in body fluid is still a challenge for early screening of cancer. Herein, by catalytic hairpin assembly as a signal amplification strategy, we designed a novel surface-enhanced Raman scattering (SERS)-lateral flow assay (LFA) strip for ultrasensitive detection of miR-21 and miR-196a-5p in non-small cell lung cancer (NSCLC) urine on a single test (T) line. 4-mercaptobenzoic acid or 5,5'-dithiobis-2-nitrobenzoic acid as Raman molecules was labeled and two hairpin DNA sequence was modified gold nanocages (GNCs) were designed as two SERS tags. Through target miRNA-triggered catalytic hairpin assembly (CHA), the double-stranded DNAs (H1-H2 complex) formed by SERS tags and the related hairpin-structured DNA sequence 2 (H2) were immobilized on a single T line of SERS-LFA strip. This generated abundant "hot spots" because of the formation of numerous H1-H2 complex thus facilitated the SERS measurement. Through this method, two kinds of miRNAs were analyzed, resulting in limits of detection of 2.08 pM and 3.31 pM for miR-21 in PBS buffer and human urine, 1.77 pM and 2.18 pM for miR-196a-5p in PBS buffer and human urine. Significantly, the SERS-LFA strip exhibited high specificity and good repeatability toward miRNAs. The whole detection time was only 30 min, which means that the high detection efficiency of the strip. The clinical feasibility of the proposed method was also evaluated by detecting the levels of miR-21 and miR-196a-5p in urine samples from NSCLC patients and healthy subjects. The developed SERS-LFA strip has wide application prospect in biomedical research, drug development and early clinical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Herein, we proposed a novel biosensor based on a high-density "hot spot" Au@SiO 2 array substrate and rolling circle amplification (RCA) strategy for the ultrasensitive detection of CpG methyltransferase (M.SssI) activity. In the presence of M.SssI, the RCA process can be triggered, causing the augmentation of the single-stranded DNA (ssDNA) at the tail of the double-stranded DNA (dsDNA), and the ssDNA can be hybridized with numerous DNA probes labeled with Raman reporters in the next steps. Afterwards, the resultant ssDNA can be modified to the Au@SiO 2 array substrate with the SERS enhancement factor of 7.49 × 10 6 . The substrate was synthesized by using a monolayer SiO 2 array to pick up the Au nanoparticle (AuNP) array and finite-difference time-domain (FDTD) simulation showed its excellent SERS effect. Particularly, the developed biosensor displayed a significant sensitivity with a broad detection range covering from 0.005 to 50 U mL -1 , and the limits of detection (LODs) in PBS buffer and human serum were 2.37 × 10 -4 U mL -1 and 2.51 × 10 -4 U mL -1 , respectively. Finally, in order to verify the feasibility of its clinical application, the serum samples of healthy subjects and breast cancer, prostate cancer, gastric cancer and cervical cancer patients were analyzed, and the reliability of the results was also confirmed by western blot (WB) experiments. Taking advantage of these merits, the proposed biosensor can be a very promising alternative tool for the detection of M.SssI activity, which is of vital importance in the early detection and prevention of tumors.

The highly sensitive and reliable detection, imaging, and monitoring of changes of intracellular caspase-3 are critical for understanding the cell apoptosis and studying the progression of caspase-3-related cervical cancer. Herein, we present a novel surface-enhanced Raman scattering (SERS) probe for the detection of caspase-3 during cervical cancer cell apoptosis, composed of Au nanoboxes modified with Nile blue A as a Raman reporter and a caspase-3-specified peptide as a molecular cross-linker. In the presence of caspase-3, the substrate peptides can be cleaved and the changed surface charge of the Au nanoboxes results in the Au nanoboxes-NBA-peptide assembling to form aggregates and a great enhancement of SERS signal. The finite-difference time-domain simulation showed that hot spots mainly located in the nanogaps of the aggregated Au nanoboxes, which in theory proved the rationality of this signal amplification method. The SERS probes exhibited excellent reproducibility and selectivity toward caspase-3. A detection limit of 0.127 fM was obtained for caspase-3, with a dynamic range from 1 fM to 1 nM. MTT assay demonstrated that the probes had no obvious cytotoxicity within a certain concentration range. HeLa cells were treated with doxorubicin to induce long-term apoptosis. Upon cellular uptake of these probes, the spatiotemporal dynamics of caspase-3 in apoptotic cells could be real-time monitored using SERS. The activity of caspase-3 increased with the prolongation of apoptosis time. The SERS results were in accordance with that of western blotting assay. This kind of probe can offer great potential for the determination of enzymatic activities in the physiological processes of cells.

Ultrasensitive detection of specific biomarkers in clinical serum is helpful for early diagnosis of cervical cancer. In this paper, a surface-enhanced Raman scattering (SERS)-based immunoassay was developed for the simultaneous determination of squamous cell carcinoma antigen (SCCA) and osteopontin (OPN) in cervical cancer serum. Au-Ag nanoshuttles (Au-AgNSs) as SERS tags and hydrophobic filter paper-based Au nanoflowers (AuNFs) as capture substrate were constructed into a sandwich structure which served as an ultrasensitive SERS-based immunoassay platform. Finite difference time domain simulation confirmed that the electromagnetic field coupled between the AuNFs had a prominent SERS signal enhancement effect, which improved the detection sensitivity. SERS mapping showed that hexadecenyl succinic anhydride hydrophobic treatment could prevent the analyte from being quickly absorbed by the filter paper and increase the retention time to be more evenly distributed on the filter paper substrate. The immunoassay platform was verified to have good selectivity and reproducibility. With this method, the detection limits of SCCA and OPN in human serum were as low as 8.628 pg/mL and 4.388 pg/mL, respectively. Finally, in order to verify the feasibility of its clinical application, the serum samples of healthy subjects; cervical intraepithelial neoplasia I (CINI), CINII, and CINIII; and cervical cancer patients were analyzed, and the reliability of the results was confirmed by enzyme-linked immunosorbent assay experiments. The constructed SERS-based immunoassay platform could be used as a clinical tool for early screening of cancers in the future.

The ability to monitor the differentiation of living stem cells is essential for understanding stem cell biology and the practical application of stem cell therapies. However, conventional methods of analyzing biomarkers related to differentiation still require a large number of cells or cell lysates. This requirement leads to the unavoidable loss of cell sources and hinders the real-time monitoring of cellular processes. In this study, we report an ultrasensitive surface-enhanced Raman scattering (SERS) method for the long-term detection and imaging of miR-144-3p in osteogenic differentiation of BMSCs, by using target miRNA-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC assembly in living cells. The finite-difference time domain method demonstrated that the electromagnetic intensities of the dimer and polymer of the GNCs were significantly enhanced compared to that of GNCs only, which theoretically confirmed the rational design of the SERS strategy. The hpDNA-conjugated GNC probes were prepared and used to recognize the target and distinguish from other miRNAs. This method enabled excellent sensitivity and high selectivity toward miR-144-3p with a limit of detection of 13.6 aM and a broad range from 100 aM to 100 pM in cell lysates. Then, we used transmission electron microscopy images, fluorescence microscopy images, and dark-field microscopy images to study the internalization of the probes in BMSCs. A Cell Counting Kit-8 experiment indicated that the probes were not cytotoxic in a certain concentration range. BMSCs were treated with an osteogenic inductor so that they would subsequently differentiate into osteocytes. Upon cellular uptake of these nanoprobes, we observed intense and time-dependent SERS responses from the important osteogenic biomarker miR-144-3p, only in BMSCs undergoing osteogenic differentiation and living undifferentiated BMSCs but not in osteoblasts. Finally, the accuracy of SERS has been proved by a quantitative real-time polymerase chain reaction experiment. The above results demonstrated that our nanoprobes are capable of long-term tracking of the dynamic expression of miR-144-3p (21 days) in the differentiating BMSCs. SERS has broad application prospects in the long-term detection of stem cell differentiation, and identification and isolation of specific cell types as well as in biomedical diagnosis.

Variations in the intracellular expression level of cancer-related microRNAs (miRNAs) are connected with worsening tumor progression. A simple, accurate, and sensitive analytical method for the imaging and detection of intracellular miRNA is still a great challenge due to the low abundance of miRNAs and the complexity of intracellular environments. In this work, target miRNA (miRNA)-mediated catalytic hairpin assembly (CHA)-induced gold nanocage (GNC)-hairpin DNA1 (hpDNA1)-hpDNA2-GNC nanostructures were designed for surface-enhanced Raman scattering (SERS) detection and imaging of the specific miR-125a-5p in the normal lung epithelial cell line (BEAS-2B cells) and lung cancer cell line (A549 cells). The finite difference time domain (FDTD) simulations showed that the polymer of GNCs possessed a much stronger electromagnetic field in nanogaps than that of single GNC, theoretically confirming the rational design of the CHA assembly strategy. Using this method, miR-125a-5p can be detected in a wide linear range with a detection limit of 43.96 aM and high selectivity over other miRNAs in vitro. Moreover, SERS imaging successfully detected and distinguished the expression levels of intracellular miR-125a-5p in BEAS-2B cells and A549 cells. The results obtained by the SERS assay were consistent with those obtained by the real-time quantitative polymerase chain reaction (qRT-PCR). This method can offer a powerful strategy for the imaging and quantitative detection of various types of biomolecules in vitro as well as in living cells.

Aim: Here, a multiplex surface-enhanced Raman scattering (SERS) based assay for simultaneous quantitation of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) was developed., Methods: SERS tags of nanostars and SERS substrates of nanobowl arrays were functionalized with labeling and capturing antibodies, respectively. In presence of antigens, SERS tags, antigens and SERS substrates formed sandwich structure., Results: The SERS-based technique showed a wide linear range from 0.5 to 100 ng/ml and detection limits were 0.41 and 0.35 ng/ml for CEA and AFP in phosphate-buffered saline buffer, respectively. Analysis results of clinical serum samples using this technique were similar to that shown in phosphate-buffered saline buffer. The LODs were 0.44 and 0.40 ng/ml for CEA and AFP, respectively. Conclusion: The precision and stability of this analysis technique were satisfactory, meanwhile, no obvious cross-reactivity could be found. What's more, it also suggested that this novel multiplex SERS-based technique could be a simple, specific, reliable, sensitive and multiplexed tool for important diagnostic and prognostic applications.

This work presents the synthesis and characterization of Au nanostars (AuNSs) and demonstrates their application as surface enhanced Raman scattering (SERS)-activity tags for cellular imaging and sensing. Nile blue A (NBA) and bovine serum albumin (BSA) were used as Raman reporter molecules and capping materials, respectively. The SERS-activity tags were tested on human lung adenocarcinoma cell (A549) and alveolar type II cell (AT II) and found to present a low level of cytotoxicity and high chemical stability. These SERS-activity tags not only can be applied in multiplexed cellular imaging, including dark field imaging, transmission electron microscopy (TEM) and SERS imaging, but also can be used for cellular sensing. The SERS spectra clearly identified cellular important components such as proteins, nucleic acids, lipids, and carbohydrates. This study also shows that endocytosis is the main channel of tags internalized in cells. The AuNSs exhibiting strong surface enhanced Raman effects are utilized in the design of an efficient, stable SERS-activity tag for intracellular applications.

Lung resident mesenchymal stem cells (LR-MSCs) are important regulators of pathophysiological processes including tissue repair and fibrosis, inflammation, angiogenesis and tumor formation. Therefore, increasing attention has focused on the functional differentiation of LR-MSCs. However, the distinction between the undifferentiated and differentiated LR-MSCs, which are closely related and morphologically similar, is difficult to achieve by conventional methods. In this study, by employing the TAT Peptide-conjugated Au nanostars (AuNSs) as an intracellular probe, we developed a method for the identification of LR-MSC differentiation by surface-enhanced Raman scattering (SERS) spectroscopy. SERS spectra were analyzed using principal component analysis (PCA) that allowed unambiguous distinction of subtypes and monitoring of component changes during cellular differentiation. Furthermore, to ascertain whether co-culture with alveolar epithelial type II (ATII) cells and incubation with transform growth factor (TGF)-β were involved in regulating the differentiation of LR-MSCs, we investigated the protein expression levels of epithelial markers and fibroblastic markers on LR-MSCs. Our results demonstrated that co-culture with ATII cells or incubation with TGF-β could induce the differentiation of LR-MSCs as confirmed by SERS analysis, a method that is capable of noninvasive characterization of and distinction between subtypes of LR-MSCs during differentiation. We have provided a new tool that may facilitate stem cell research., (Copyright © 2015 Elsevier Ltd. All rights reserved.)