Metal-Graphene Hybrid Terahertz Metasurfaces for Circulating Tumor DNA Detection Based on Dual Signal Amplification.

Terahertz (THz) spectroscopy has impressive capability for label-free biosensing, but its utility in clinical laboratories is rarely reported due to often unsatisfactory detection performances. Here, we fabricated metal-graphene hybrid THz metasurfaces (MSs) for the sensitive and enzyme-free detection of circulating tumor DNA (ctDNA) in pancreatic cancer plasma samples. The feasibility and mechanism of the enhanced effects of a graphene bridge across the MS and amplified by gold nanoparticles (AuNPs) were investigated experimentally and theoretically. The AuNPs serve to boost charge injection in the graphene film and result in producing a remarkable change in the graded transmissivity index to THz radiation of the MS resonators. Assay design utilizes this feature and a cascade hybridization chain reaction initiated on magnetic beads in the presence of target ctDNA to achieve dual signal amplification (chemical and optical). In addition to demonstrating subfemtomolar detection sensitivity and single-nucleotide mismatch selectivity, the proposed method showed remarkable capability to discriminate between pancreatic cancer patients and healthy individuals by recognizing and quantifying targeted ctDNAs. The introduction of graphene to the metasurface produces an improved sensitivity of 2 orders of magnitude for ctDNA detection. This is the first study to report the combined application of graphene and AuNPs in biosensing by THz spectroscopic resonators and provides a combined identification scheme to detect and discriminate different biological analytes, including nucleic acids, proteins, and various biomarkers.

Rapid and sensitive detection of Staphylococcus aureus using a THz metamaterial biosensor based on aptamer-functionalized Fe3O4@Au nanocomposites.

Staphylococcus aureus (S. aureus) poses a serious threat to global public health, necessitating the establishment of rapid and simple tools for its accurate identification. Herein, we developed a terahertz (THz) metamaterial biosensor based on aptamer-functionalized Fe3O4@Au nanocomposites for quantitative S. aureus assays in different clinical samples. Fe3O4@Au@Cys@Apt has the dual advantages of magnetism and a high refractive index in the THz range and was used to rapidly separate and enrich target bacteria in a complex environmental solution. Furthermore, conjugation to the nanocomposites significantly increased the resonance frequency shift of the THz metamaterial after target loading. Our results showed that the shifts in the metamaterial resonance frequency were linearly related to S. aureus concentrations ranging from 1.0 × 103 to 1.0 × 107 CFU/mL, with a detection limit of 4.78 × 102 CFU/mL. The biosensor was further applied to S. aureus detection in spiked human urine and blood with satisfactory recoveries (82.4-109.6%). Our approach also demonstrated strong concordance with traditional plate counting (R2 = 0.99306) while significantly lowering the analysis time from 24 h to <1 h. In conclusion, the proposed biosensor can not only perform culture-free and extraction-free detection of target bacteria but can also be easily extended to the determination of other pathogenic bacteria, rendering it suitable for various bacteria-related disease diagnoses.

A Novel Optical Fiber Terahertz Biosensor Based on Anti-Resonance for The Rapid and Nondestructive Detection of Tumor Cells.

The sensitive and accurate detection of tumor cells is essential for successful cancer therapy and improving cancer survival rates. However, current tumor cell detection technologies have some limitations for clinical applications due to their complexity, low specificity, and high cost. Herein, we describe the design of a terahertz anti-resonance hollow core fiber (THz AR-HCF) biosensor that can be used for tumor cell detection. Through simulation and experimental comparisons, the low-loss property of the THz AR-HCF was verified, and the most suitable fiber out of multiple THz AR-HCFs was selected for biosensing applications. By measuring different cell numbers and different types of tumor cells, a good linear relationship between THz transmittance and the numbers of cells between 10 and 106 was found. Meanwhile, different types of tumor cells can be distinguished by comparing THz transmission spectra, indicating that the biosensor has high sensitivity and specificity for tumor cell detection. The biosensor only required a small amount of sample (as low as 100 µL), and it enables label-free and nondestructive quantitative detection. Our flow cytometry results showed that the cell viability was as high as 98.5 ± 0.26% after the whole assay process, and there was no statistically significant difference compared with the negative control. This study demonstrates that the proposed THz AR-HCF biosensor has great potential for the highly sensitive, label-free, and nondestructive detection of circulating tumor cells in clinical samples.

Flexible Terahertz Metamaterial Biosensor for Ultra-Sensitive Detection of Hepatitis B Viral DNA Based on the Metal-Enhanced Sandwich Assay.

The high sensitivity and specificity of terahertz (THz) biosensing are both promising and challenging in DNA sample detection. This study produced and refined a flexible THz MM biosensor for ultrasensitive detection of HBV in clinical serum samples based on a gold magnetic nanoparticle-mediated rolling circle amplification (GMNPs@RCA) sandwich assay under isothermal conditions. Typically, solid-phase RCA reactions mediated by circular padlock probes (PLPs) are triggered under isothermal conditions in the presence of HBV DNA, resulting in long single-stranded DNA (ssDNA) with high fidelity and specificity. Then, the resultant ssDNA was conjugated with detection probes (DPs) immobilized on gold nanoparticles (DP@AuNPs) to form GMNPs-RCA-AuNPs sandwich complexes. The HBV DNA concentrations were quantified by introducing GMNPs-RCA-AuNPs complexes into the metasurface of a flexible THz metamaterial-based biosensor chip and resulting in a red shift of the resonance peak of the THz metamaterials. This biosensor can lead to highly specific and sensitive detection with one-base mismatch discrimination and a limit of detection (LOD) down to 1.27E + 02 IU/ml of HBV DNA from clinical serum samples. The HBV DNA concentration was linearly correlated with the frequency shift of the THz metamaterials within the range of 1.27E + 02∼1.27E + 07 IU/ml, illustrating the applicability and accuracy of our assay in real clinical samples. This strategy constitutes a promising THz sensing method to identify virus DNA. In the future, it is hoped it can assist with pathogen identification and clinical diagnosis.

Evaluation of classification ability of logistic regression model on SERS data of miRNAs.

Logistic regression (LR) is a supervised multiple linear regression model, which uses linear weighted calculation for input to obtain weight coefficients of model. The surface enhanced Raman spectroscopy (SERS) technology greatly enhances the Raman signal of analyte. LR model was used to analyze the data of seven types of pancreatic cancer-related miRNAs obtained from commercial SERS substrate. The classification ability of the model on such data was observed under the configurations of different key parameters (classification mode, regularization method and loss function optimization way), and the effect of the two types of data formats were also evaluated. The results showed that though LR model used to classify this data did not perform well as expected, miRNA-191 and miRNA-4306 still had high recalls (sensitivity), which laid a theoretical foundation for the purpose of using LR model to identify these two miRNAs to jointly diagnose of pancreatic cancer at miRNA level.

THz-ATR Spectroscopy Integrated with Species Recognition Based on Multi-Classifier Voting for Automated Clinical Microbial Identification.

The demand for rapid and accurate identification of microorganisms is growing due to considerable importance in all areas related to public health and safety. Here, we demonstrate a rapid and label-free strategy for the identification of microorganisms by integrating terahertz-attenuated total reflection (THz-ATR) spectroscopy with an automated recognition method based on multi-classifier voting. Our results show that 13 standard microbial strains can be classified into three different groups of microorganisms (Gram-positive bacteria, Gram-negative bacteria, and fungi) by THz-ATR spectroscopy. To detect clinical microbial strains with better differentiation that accounts for their greater sample heterogeneity, an automated recognition algorithm is proposed based on multi-classifier voting. It uses three types of machine learning classifiers to identify five different groups of clinical microbial strains. The results demonstrate that common microorganisms, once time-consuming to distinguish by traditional microbial identification methods, can be rapidly and accurately recognized using THz-ATR spectra in minutes. The proposed automatic recognition method is optimized by a spectroscopic feature selection algorithm designed to identify the optimal diagnostic indicator, and the combination of different machine learning classifiers with a voting scheme. The total diagnostic accuracy reaches 80.77% (as high as 99.6% for Enterococcus faecalis) for 1123 isolates from clinical samples of sputum, blood, urine, and feces. This strategy demonstrates that THz spectroscopy integrated with an automatic recognition method based on multi-classifier voting significantly improves the accuracy of spectral analysis, thereby presenting a new method for true label-free identification of clinical microorganisms with high efficiency.

TNF-α Antagonizes the Effect of Leptin on Insulin Secretion through FOXO1-Dependent Transcriptional Suppression of LepRb in INS-1 Cells.

Proinflammatory cytokines play a causal role in the development of hyperinsulinemia and T2MD. FOXO1, a transcription factor which is known to enhance proinflammation, was recently shown to be involved in obesity-induced ß cell dysfunction. However, molecular mechanisms for the association remained elusive. In this study, we first found that both leptin (10 nM) and TNF-α (20 ng/ml) significantly inhibited glucose-stimulated insulin secretion (GSIS) of INS-1E cells. When in combination, the GSIS function of INS-1E cells was significantly increased compared with that of the leptin alone treatment, indicating that TNF-α attenuated the inhibiting effect of leptin on GSIS of INS-1E cells. Similarly, we found that TNF-α has the same inhibitory effect on leptin in regulating insulin synthesis and secretion, and the survival and apoptosis of insulin cells. Further studies showed that TNF-α blocks leptin pathway by reducing the expression of leptin receptor (LepRb, also called OBRb) and inhibiting the activation of STAT3, a key molecule involved in the leptin signaling pathway in INS-1E cells. Besides, the downregulated expression of phosphorylated FOXO1 was found to be involved in the possible mechanism of TNF-α. Overexpression of constitutively active FOXO1 markedly aggravated the LepRb reduction by TNF-α treatment of INS-1E cells, and the endogenous FOXO1 knockdown abolished the effect of TNF-α on INS-1E cells. Furthermore, we have proved that FOXO1 could directly bind to the promoter of LepRb as a negative transcription regulator. Taken together, the results of this study reveal that TNF-α-induced LepRb downregulated in pancreatic ß cells and demonstrate that transcriptional reduction of FOXO1 might be the primary mechanism underlying TNF-α promoting INS-1E leptin resistance and ß cell dysfunction. Conclusions. Our current studies based on INS-1E cells in vitro indicate that the inflammatory factor TNF-α plays an important role in the development of INS-1E leptin resistance and glucose metabolism disorders, probably through FOXO1-induced transcription reduction of LepRb promoter in pancreatic ß cells, and FOXO1 may be a novel target for treating ß cell dysfunction in obesity-induced hyperinsulinemia and T2DM.

Streptavidin-functionalized terahertz metamaterials for attomolar exosomal microRNA assay in pancreatic cancer based on duplex-specific nuclease-triggered rolling circle amplification.

Exosomal microRNA (miRNA) is a promising non-invasive biomarker for liquid biopsies. Herein, we fabricated a terahertz (THz) metamaterial biosensor that comprises an array of gold (Au) discs surrounded by annular grooves for exosomal miRNA assays based on duplex-specific nuclease (DSN)-triggered rolling circle amplification (RCA). In this strategy, the target miRNA is captured by a probe P0 immobilized on magnetic beads (MBs); it then repeatedly releases a primer P1 under the action of DSN, which acts as a highly specific initiator of the subsequent RCA step utilizing biotin-dUTP. After target recycling and nucleic acid amplification, the biotinylated amplification products were captured by the streptavidin (SA)-functionalized THz metamaterials, and further conjugated to SA-modified AuNPs that permit formation of a trimeric complex of SA-biotinylated RCA products-AuNP. The complex population scales with the starting concentration of the target miR-21, resulting in a red shift of the resonance peak of the THz metamaterials. This biosensor can lead to highly specific and sensitive detection with one-base mismatch discrimination and a limit of detection (LOD) down to 84 aM. Significant distinctions are seen in the frequency shifts for exosomal miR-21 quantitation in clinical plasma samples between pancreatic cancer patients and healthy controls. The frequency shifts of the THz metamaterials are consistent versus the reverse transcription-polymerase chain reaction (RT-PCR) results, illustrating the applicability and accuracy of our assay in real clinical samples.

A novel quantification platform for point-of-care testing of circulating MicroRNAs based on allosteric spherical nanoprobe.

MiRNA-150, a gene regulator that has been revealed to be abnormal expression in non-small cell lung cancer (NSCLC), can be regarded as a serum indicator for diagnosis and monitoring of NSCLC. Herein, a new sort of nanoprobe, termed allosteric spherical nanoprobe, was first developed to sense miRNA-150. Compared with conventional hairpin, this new nanoprobe possesses more enrichment capacity and reaction cross section. Structurally, it consists of magnetic nanoparticles and dual-hairpin. In the absence of miRNA-150, the spherical nanoprobes form hairpin structure through DNA self-assembly, which could promote the Förster resonance energy transfer (FRET) of fluorophore (FAM) and quencher (BHQ1) nearby. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active "Y" structure which separated fluorophore and quencher to yield "signal on" fluorescence. In the manner of multipoint fluorescence detection, the target-bound allosteric spherical nanoprobe could provide high detection sensitivity with a linear range of 100 fM to 10 nM and a detection limit of 38 fM. More importantly, the proposed method can distinguish the expression of serum miRNA-150 among NSCLC patients and healthy people. Finally, we hoped that the potential bioanalytical application of this nanoprobe strategy will pave the way for point-of-care testing (POCT).