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We report a surprising discovery that Prussian blue (PB) can be employed as a highly sensitive and background-free resonant Raman reporter. Conventional Raman reporters show multiple spectral bands in the fingerprint region, which are generally overlapped with those from dominant endogenous biomolecules, and are thus difficult to be separated. Herein, we found that PB only possesses a strong and sharp single-band in the cellular Raman-silent region, where no Raman signals from biological species were observed. Therefore, the Raman spectra from PB and endogenous biomolecules are completely resolved without resorting to complicated spectral unmixing. Moreover, PB holds a strong UV-vis absorption band between 500 and 900 nm, which is resonant with the incident detection lasers, providing extremely high sensitivity. Through assembly of PB onto plasmonic cores, a new surface-enhanced resonance Raman scattering (SERRS) probe was achieved with a high signal-to-background ratio (SBR). We demonstrated the performance of the PB-based SERRS tags for high-sensitivity immunoassay and cancer cell imaging. [ABSTRACT FROM AUTHOR]

Pyroptosis has been reported to improve the antitumor effect by evoking a more intense immune response and a therapeutic effect. For phototherapy, several photosensitizers have been found to initiate pyroptosis. However, the effect of pyroptosis associated with apoptosis in enhancing the antitumor therapy needs sufficient characterization, especially under long-term treatment. As a NIR photosensitizer, heptamethine cyanines have been discovered for anticancer phototherapy for deep tissue penetration and inherent tumor-targeted capability. However, they are not quite stable for long-term performance. To investigate the effect of pyroptosis along with apoptosis on the anticancer immune responses and phototherapy, here, we chemically modulate the cyanine IR780 to regulate hydrophobicity, stability, and intracellular targeting. Two photosensitizers, T780T-TPP and T780T-TPP-C 12 , were finally optimized and showed excellent photostability with high photothermal conversion efficiency. Although the cellular uptake of the two molecules was both mediated by OATP transporters, T780T-TPP induced tumor cell death via pyroptosis and apoptosis and accumulated in tumor accumulation, while T780T-TPP-C 12 was prone to accumulate in the liver. Ultimately, via one injection-multiple irradiation treatment protocol, T780T-TPP displayed a significant antitumor effect, even against the growth of large tumors (200 mm 3 ).

Phenotypic plasticity is an emerging paradigm for providing biological and clinical insights into cancer initiation, progression, and resistance to therapy. However, it is a great challenge to track phenotypic information on live cells with high levels of sensitivity, specificity, and simplicity, when a specific cancer-cell subset is being targeted. In this work, we have successfully achieved cascade assembly of nanoparticles on the surface of specific cancer cells by designing a dual-aptamer-weaved molecular AND logic system. Taking advantage of spatial addressability, precise controllability, and targeting recognition of the nanostructure assemblies, we can precisely label the target-cell subset in a large population of similar cells and rapidly obtain phenotypic information in response to the surface changes of captured cancer cells. Without sophisticated instruments, we can know the phenotypic information on HepG2 cells in whole blood with a high level of sensitivity and rapid naked-eye tracking of on-cell phenotype changes of HepG2 cells undergoing epithelial-mesenchymal transition.

Glioblastoma (GBM) is one of the most fatal tumors in the brain, and its early diagnosis remains technically challenging due to the complex repertoires of oncogenic alterations and blood-brain barrier (BBB). GBM-derived specific exosomes can cross the BBB and circulate in body fluids, so they can be noninvasive biomarkers for the early diagnosis of GBM. Herein, we propose a sensitive and label-free electrochemical biosensor designed by using Zr-based metal-organic frameworks (Zr-MOFs) for the detection of GBM-derived exosomes with practical application. In the design, a peptide ligand can specifically bind with human epidermal growth factor receptor (EGFR) and EGFR variant (v) III mutation (EGFRvIII), which are overexpressed on the GBM-derived exosomes. Meanwhile, Zr-MOFs encapsulated with methylene blue can absorb on the surface of the exosomes due to the interaction between Zr 4+ and the intrinsic phosphate groups outside of exosomes. Consequently, the concentration of exosomes can be directly quantified by monitoring the electroactive molecules inside MOFs, ranging from 9.5 × 10 3 to 1.9 × 10 7 particles/μL with the detection of limit of 7.83 × 10 3 particles/μL. Furthermore, this proposed biosensor can distinguish GBM patients from healthy groups, demonstrating the great prospect for early clinical diagnosis.

Peptidylarginine deiminase 4 (PAD4) is the only isoform of PADs located within the cell nucleus, which has been known to be related to several human diseases. In this work, we have proposed an electrochemical method for the assay of endogenous PAD4 activities as well as the studies of PAD4 inhibitors by making use of the supramolecular chemistry-assisted signal labeling. Specifically, peptide probes P1 and P2, which separately contain cysteine residues and tripeptides FGG (Phe-Gly-Gly), can be self-assembled onto the surface of the gold electrode and silver nanoparticles, respectively. In the meantime, the peptide probes can be connected together through cucurbit[8]uril-mediated host-guest interaction. Nevertheless, after trypsin-catalyzed digestion, FGG at the N-terminal of P1 will be removed from the electrode surface, thereby inhibiting the connection of P1 and P2. Since PAD4 catalyzes the citrullination of arginine residue within P1, trypsin-catalyzed digestion of P1 can be prohibited by the addition of PAD4. Consequently, an obvious change of the electrochemical response can be obtained from the silver nanoparticles (AgNPs) immobilized on the electrode surface. Experimental results have shown that our method can display an improved sensitivity and specificity for both PAD4 assay and inhibitor screening, which may effectively trace endogenous PAD4 and the inhibitors in the cancer cells. Therefore, our method may have great potential for the diagnosis and treatment of PAD4-related diseases in the future.

Aberrant methylation by DNA transferase is associated with cancer initiation and progression. For high-throughput screening of DNA methyltransferase (MTase) activity and its inhibitors, a novel chemiluminescence immunoassay (CLIA) was established to detect S-adenosylhomocysteine (SAH), the product of S-adenosylmethionine (SAM) transmethylation reactions. We synthesized two kinds of immunogens for SAH and characterized the polyclonal antibodies in each group. The antibody with higher titer was used to develop a competitive CLIA for SAH, in which SAH in samples would compete with SAH coated on microplate in binding with SAH antibodies. Successively, horseradish peroxidase labeled goat antirabbit IgG (HRP-IgG) was conjugated with SAH antibodies on the microplate. In substrate solution containing luminol and H2O2, HRP-IgG catalyzed luminol oxidation by H2O2, generating a high chemiluminescence signal. The method could detect as low as 9.8 ng mL(-1) SAH with little cross-reaction (3.8%) to SAM. Since higher DNA MTase activity leads to more production of SAH, a correlation between the chemiluminescence intensity and DNA MTase activity was obtained in the range from 0.1 to 8.0 U/mL of DNA MTase. The inhibition study showed that, in the presence of SAM as methyl donor, Lomeguatrib, 5-Azacytidine, and 5-Aza-2'-deoxycytidine could inhibit the DNA MTase activity with IC50 values of 40.57 nM, 2.26 μM, and 0.48 μM, respectively. These results are consistent with the published studies. The proposed assay does not depend on recognizing methylated cytosines in oligonucleotides (methyl acceptor) and showed the potential as an accessible platform for sensitive detection of DNA MTase activity and screening its inhibitors.

Detection of metastatic activity before the onset of the actual metastasis can be a promising method to combat metastasis, the foremost cause of death in cancer. Therefore, in this work, we have developed an assay method for the detection of metastatic tumor cells in primary tumor, by using a protein of the metastatic cell signaling as the biomarker. In this assay, a peptide-based probe targeting the marker protein and a sensitive nanoparticle doped graphene nanolabel are combined to enable the detection of metastatic cells. Consequently, the metastatic cells can be specifically detected and discriminated from primary tumor cells. By applying this assay method to clinical samples of primary tumor, a low amount of metastatic activity can be detected in the tumor sites, which may suggest the activity of local metastatic transformation. So, these results may point to the prospect of using the proposed method for controlling metastatic cancer.

As one of the rarely allowable azo dyes, ponceau 4R can be added in some foods in some countries. However, it is necessary to develop a credible and rapid analytical method for its monitoring, because of its potentially harmful risk. The hapten of ponceau 4R was first designed and synthesized by introducing a primary amine group into the structure of ponceau 4R. Based on the well-prepared hapten, the immunogen of ponceau 4R was prepared using glutaraldehyde to link ponceau 4R to the carrier protein. The triggered polyclonal antibody was obtained and tested by ELISA to optimize the proper dilution. An icELISA was developed for ponceau 4R, and the IC50 of the method is 36.82 ng/mL. The limit of detection is 0.80 ng/mL, and the linear range is 1-10000 ng/mL. Five selected structural analogues have no cross-reactivity with the anti-ponceau 4R polyclonal antibody (<0.3). In three food samples (grape juice, carbonated beverage, and RIO cocktail), the assay exhibits good stability and reproducibility with a recovery range of 93.87-103.77%, and the intra- and interassay coefficients of variation were <11.73%. The results indicate that the proposed icELISA is sensitive, accurate, specific, and simple, which provides an alternative for the detection of ponceau 4R in foods.

In this work, a novel method for a protein assay is proposed which uses the specific protein-binding peptide of the target protein and sequence-specific DNA to interact with the target as the capture and detection probe, respectively. Meanwhile, since the DNA sequence can be coupled with gold nanoparticles to amplify the signal readout, a sensitive and easily operated method for protein assay is developed. We have also employed a transcription factor named as cysteine-rich intestinal protein 1 (CRIP1), which has been identified as an ideal biomarker for staging of breast cancer, as the model protein for this study. With the proposed method, CRIP1 can be determined in a linear range from 1.25 to 10.13 ng/mL, with a detection limit of 1.25 ng/mL. Furthermore, the proposed method can be directly used to assay CRIP1 in tissue samples. Owing to its desirable sensitivity, excellent reproducibility, and high selectivity, the proposed method may hold great potential in clinical practice in the future.

An indirect immunoassay for the determination of vitamin B2 in food samples and vitamin tablets was developed. A carbodiimide-modified active ester method was used to synthesize the immunogen for vitamin B2. The coupling ratio of vitamin B2 to carrier protein in immunogen was 19.98:1. The titer of the polyclonal antibody was 1:64000, and the antibody showed high specificity in the presence of vitamin B2 photolytic products and other B group vitamins. The immunoassay showed detection limits (LODs) of 1.07 ng/mL in PBS, 24.6 ng/g in vitamin drink, and 0.50 mg/kg in milk powder. Recovery was 99.58-110.91% in milk powder and 70.20-100.5% in vitamin drink. Vitamin B2 samples were analyzed by high-pressure liquid chromatography (HPLC) and the immunoassay, and results showed good agreement. Finally, this method was applied to detect vitamin B2 in commercial milk powder and vitamin tablets, and the detected amount correlated well with the labeled amount.

Protein-binding peptide is recently recognized as an effective artificial affinity reagent for protein assays. However, its application is hampered by the limited choices of available signal readout methods. Herein, we report a general electrochemical signal readout method for protein-binding peptides exploiting the host-guest chemistry of cucurbituril. Via the formation of supermolecules among cucurbituril, electrochemical reporter, and the peptide, a protein-binding peptide can be noncovalently coupled with the electrochemical reporter. To assay the target protein, the protein-binding peptides are first self-assembled in the sensing layer, and after the capturing of the target protein, a portion of the peptides become protein-bound. The protein-free peptides are then coupled with the electrochemical reporter to yield a signal readout inversely proportional to the amount of the captured target proteins. Since the only requirement of supermolecule formation is the incorporation of aromatic amino acids in the peptide sequence, this strategy is universally applicable to many protein-binding peptides. The generality and target specificity of the proposed method are successfully demonstrated in the assays of two kinds of target proteins: tumor necrosis factor-α and amyloid β 1-42 soluble oligomer, respectively. The feasibility of our method is also tested in the monitoring of tumor necrosis factor-α secretion activity of HL-60 cells. These results indicate that our method can have great use in protein detection in the future.

Based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe 1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe 2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe(CN)(6)](3-/4-) becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from 0.3 to 15 ng/mL with a detection limit of 0.19 ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future.