Low-abundance proteins-based label-free SERS approach for high precision detection of liver cancer with different stages.

BACKGROUND: Surface-enhanced Raman scattering (SERS) technology have unique advantages of rapid, simple, and highly sensitive in the detection of serum, it can be used for the detection of liver cancer. However, some protein biomarkers in body fluids are often present at ultra-low concentrations and severely interfered with by the high-abundance proteins (HAPs), which will affect the detection of specificity and accuracy in cancer screening based on the SERS immunoassay. Clearly, there is a need for an unlabeled SERS method based on low abundance proteins, which is rapid, noninvasive, and capable of high precision detection and screening of liver cancer. RESULTS: Serum samples were collected from 60 patients with liver cancer (27 patients with stage T1 and T2 liver cancer, 33 patients with stage T3 and T4 liver cancer) and 40 healthy volunteers. Herein, immunoglobulin and albumin were separated by immune sorption and Cohn ethanol fractionation. Then, the low abundance protein (LAPs) was enriched, and high-quality SERS spectral signals were detected and obtained. Finally, combined with the principal component analysis-linear discriminant analysis (PCA-LDA) algorithm, the SERS spectrum of early liver cancer (T1-T2) and advanced liver cancer (T3-T4) could be well distinguished from normal people, and the accuracy rate was 98.5% and 100%, respectively. Moreover, SERS technology based on serum LAPs extraction combined with the partial least square-support vector machine (PLS-SVM) successfully realized the classification and prediction of normal volunteers and liver cancer patients with different tumor (T) stages, and the diagnostic accuracy of PLS-SVM reached 87.5% in the unknown testing set. SIGNIFICANCE: The experimental results show that the serum LAPs SERS detection combined with multivariate statistical algorithms can be used for effectively distinguishing liver cancer patients from healthy volunteers, and even achieved the screening of early liver cancer with high accuracy (T1 and T2 stage). These results showed that serum LAPs SERS detection combined with a multivariate statistical diagnostic algorithm has certain application potential in early cancer screening.

Quantitative and direct serum albumin detection by label-free SERS using tunable hydroxyapatite nanostructure for prostate cancer detection.

With the aid of good biocompatibility and stability with hydroxyapatite (HAp) in protein separation and adsorption fields, we developed a novel extraction-isolation albumin analysis method by relying on the specific adsorption capacity of HAp, combining with surface-enhanced Raman spectroscopy (SERS) for prostate cancer screening. Two different nanostructures of HAp particles, including the HAp flower and HAp sphere, were synthesized with a hydrothermal method, and the targeted binding and extraction abilities of serum albumin of these two HAp particles were compared. By changing the morphology of the nanostructure, the albumin-adsorption capacity of HAp varied significantly. Compared with spherical HAp particles, HAp flower particles have more albumin binding sites per unit area. Thus, the HAp flower displayed the superior capacity for adsorption-release of albumin, which was further employed for clinical prostate cancer screening. Based on the superior adsorption-extraction ability of albumin of HAp flower, serum albumin was adsorbed and extracted by HAp flower from serum samples of prostate cancer patients (n = 30) and healthy volunteers (n = 30), and mixed with silver colloids to perform SERS spectral analysis. The partial least square-support vector machines (PLS-SVM) model is used to analyze the obtained serum albumin SERS spectra and establish the diagnostic model, the diagnostic accuracy was up to 95.00% for differentiating the normal volunteer from prostate patient groups. The results demonstrate that the PLS-SVM model provides superior performance in the classification of a prostate cancer diagnosis. Due to the advantages of simplicity and rapidness, the HAp flower-adsorbed-released albumin combined with SERS was expected to become a promising tool for prostate cancer detection.

Label-free detection of bladder cancer and kidney cancer plasma based on SERS and multivariate statistical algorithm.

In this study, we mainly aimed to investigate the diagnostic potential of surface-enhanced Raman spectroscopy for bladder cancer and kidney cancer which are the most common cancers of the urinary system, and evaluate the classification ability of three statistical algorithms: principal component analysis-linear discriminate analysis (PCA-LDA), partial least square-random forest (PLS-RF), and partial least square-support vector machine (PLS-SVM). The plasma of 26 bladder cancer patients, 38 kidney cancer patients and 39 normal subjects was mixed with the same volume of silver nanoparticles, respectively, and then high-quality SERS signal was obtained. The SERS spectra in the range of 400-1800 cm-1 were compared and analyzed. There were some significant differences in SERS peak intensity, which may reflect the changes in the content of some biomacromolecules in the plasma of cancer patients. Based on the three algorithms of PCA-LDA, PLS-RF and PLS-SVM, the classification accuracy of SERS spectra of plasma from cancer patients and normal subjects was 98.1%, 100% and 100%, respectively. In addition, the classification accuracy of the three diagnostic algorithms to classify the SERS spectra of bladder cancer and kidney cancer was 81.3%, 91.7%, and 98.4%, respectively. This exploratory work demonstrates that SERS combined with PLS-SVM algorithm has superior performance for clinical screening of bladder cancer and kidney cancer through peripheral plasma.

Study on degranulation of mast cells under C48/80 treatment by electroporation-assisted and ultrasound-assisted surface-enhanced Raman spectrascopy.

Both electroporation-assisted and ultrasound-assisted delivery methods can rapidly deliver nanoparticles into living cells for surface-enhanced Raman scattering (SERS) detection, but these two methods have never been compared. In this study, electroporation-assisted SERS and ultrasound-assisted SERS were employed to detect the biochemical changes of degranulated mast cells induced by mast cell stimulator (C48/80). The results showed that the cell damage of electroporation based on controllable electric pulse was smaller than that of ultrasound based on cavitation. Transmission electron microscope images of cells indicated that the nanoparticles delivered by electroporation were mainly distributed in the cytoplasm, while ultrasound could transport nanoparticles to the cytoplasm and nucleus. Therefore, electroporation-assisted SERS mainly detects the biochemical information of cytoplasm, while ultrasound-assisted SERS gets more spectral signals of nucleic acid. Both methods can obtain high quality SERS signal of cells. With drug treatment, the SERS peak intensity of 733 cm-1 attributed to phosphatidylserine decreased significantly, which may be due to the activation of mast cell degranulation pathway stimulated by C48/80 agonist, resulting in a large amount of intracellular serine being used to synthesize tryptase, while the production of phosphatidylserine decreased. Further, based on principal component analysis and linear discriminant analysis (PCA-LDA approach), ultrasound-assisted SERS could achieve better sensitivity, specificity and accuracy in the discrimination and identification of drug-treated degranulated mast cells than electroporation assisted SERS. This exploratory work is helpful to realize the real-time dynamic SERS detection of intracellular biochemical components, and it also has great potential in intracellular SERS analysis, such as the cytotoxicity assay of anti-tumor drugs or cancer cell screening.

Label-free surface enhanced Raman spectroscopy analysis of blood serum via coffee ring effect for accurate diagnosis of cancers.

Surface-enhanced Raman spectroscopy (SERS) is considered as an ultrasensitive, non-invasive as well as rapid detection technology for cancer diagnosis. In this study, we developed a novel blood serum analysis strategy using coffee ring effect-assisted label-free SERS for different types of cancer screening. Additionally, the pretreated Ag nanoparticles (Ag NPs) were mixed with the serum from liver cancer patients (n = 40), prostate cancer patients (n = 32) and healthy volunteers (n = 30) for SERS measurement. The droplets of Ag NPs-serum mixture formed the coffee ring on the peripheral after air-drying, and thus extremely enhancing Raman signal and ensuring the stability and reliability of SERS detection. Partial least square (PLS) and support vector machine (SVM) algorithms were utilized to establish the diagnosis model for SERS spectra data classifying, yielding the high diagnostic accuracy of 98.04% for normal group and two types of cancers simultaneously distinguishing. More importantly, for the unknown testing set, an ideal diagnostic accuracy of 100% could be achieved by PLS-SVM algorithm for differentiating cancers from the normal group. The results from this exploratory work demonstrate that serum SERS detection combined with PLS-SVM diagnostic algorithm and coffee ring effect has great potential for the noninvasive and label-free detection of cancer.

Quantitative detection of crystal violet using a surface-enhanced Raman scattering based on a flower-like HAp/Ag nanocomposite.

Herein, we proposed a simple one-pot sol-thermal strategy to prepare a highly sensitive and reproducible SERS substrate. The silver-doped hydroxyapatite nanocomposite (HAp/Ag) could suppress the oxidation of silver nanoparticles, which endow the SERS substrate with good stability and reproducibility. Due to the strong interaction between the HAp/Ag substrate and the analytes, a stronger Raman signal generated during the process of SERS detection. In particular, the HAp/Ag substrate enabled the determination of rhodamine 6G (R6G) and crystal violet (CV), and the limits of detection (LOD) were low at 10-6 M and 10-5 M, respectively. In addition, the HAp/Ag substrate could be used for the quantitative analysis of CV in wastewater with a good linear relationship between 10-2 and 10-5 M. In this context, the HAp/Ag substrate combines the superior properties of both Ag NPs and HAp particles, providing a potential method for monitoring the environment and building a convenient SERS platform to detect pollutants in wastewater.

Label-free determination of liver cancer stages using surface-enhanced Raman scattering coupled with preferential adsorption of hydroxyapatite microspheres.

Here, we explored a label-free albumin targeted analysis method by utilizing hydroxyapatite (HAp) to adsorb-release serum albumin, in conjunction with surface-enhanced Raman scattering (SERS) for screening liver cancer (LC) at different tumor (T) stages. Excitingly, albumin can be preferentially adsorbed by HAp as compared with other serum proteins. Moreover, we developed a novel strategy using a high concentration of PO43- solution as the albumin-release agent. This method overcomes the shortcomings of the traditional purification technology of serum albumin, which requires acid to release protein, and ensures that the structure and properties of albumin are not damaged. The SERS spectra of serum albumin obtained from three sample groups were analyzed to verify the feasibility of this new method: healthy volunteers (n = 35), LC patients with T1 stage (n = 25) and LC patients with T2-T4 stage (n = 23). Furthermore, principal component analysis (PCA) combined with linear discriminant analysis (LDA) was employed to classify the early T (T1) stage LC vs. normal group and advanced T (T2-T4) stage LC vs. normal group, yielding high diagnostic accuracies of 90.00% and 96.55%, respectively, which showed a 10% improvement in diagnostic accuracy for the early stage detection of cancer as compared with previous studies. The results of this exploratory work demonstrated that HAp-adsorbed-released serum albumin combined with SERS analysis has great potential for label-free, noninvasive and sensitive detection of different T stages of liver cancer.

Label-free diagnosis of breast cancer based on serum protein purification assisted surface-enhanced Raman spectroscopy.

Serum protein is generally used to assess the severity of disease, as well as cancer progression and prognosis. Herein, a simple and rapid serum proteins analysis method combined with surface-enhanced Raman spectroscopy (SERS) technology was applied for breast cancer detection. The cellulose acetate membrane (CA) was employed to extract human serum proteins from 30 breast cancer patients and 45 healthy volunteers and then extracted proteins were mixed with silver nanoparticles for SERS measurement. Additionally, we also mainly assessed the use of different ratios of proteins-silver nanoparticles (Ag NPs) mixture to generate maximum SERS signal for clinical samples detection. Two multivariate statistical analyses, principal component analysis-linear discriminate analysis (PCA-LDA) and partial least square-support vector machines (PLS-SVM) were used to analyze the obtained serum protein SERS spectra and establish the diagnostic model. The results demonstrate that the PLS-SVM model provides superior performance in the classification of breast cancer diagnosis compared with PCA-LDA. This exploratory work demonstrates that the label-free SERS analysis technique combined with CA membrane purified serum proteins has great potential for breast cancer diagnosis.

A microsphere nanoparticle based-serum albumin targeted adsorption coupled with surface-enhanced Raman scattering for breast cancer detection.

The serum albumin level is inseparable associated with survival in patients with breast cancer, and simultaneously serve as a good indicator of prognosis of cancer. Here, we proposed a novel extraction-isolation analysis method of albumin for breast cancer detection utilizing hydroxyapatite particles (HAp) to targeted adsorb albumin from serum relying on its specific adsorption capacity. An ideal protein-release reagent was used for isolating albumin from the surface of HAp, and meanwhile ensuring that the structure and property of albumin was not suffered damage. The surface-enhanced Raman scattering (SERS) signal of extracted albumin was obtained, and partial least squares (PLS) and linear discriminant analysis (LDA) analysis approach were employed to analyze SERS spectra data, with the aim to assess the capability of HAp method for identifying breast cancer, yielding an ideal diagnostic accuracy of 98.6%, demonstrating promising potential as a non-invasive and sensitive nanotechnology for breast cancer screening.

A Putative C2H2 Transcription Factor CgTF6, Controlled by CgTF1, Negatively Regulates Chaetoglobosin A Biosynthesis in Chaetomium globosum.

Gα signaling pathway as well as the global regulator LaeA were demonstrated to positively regulate the biosynthesis of chaetoglobosin A (ChA), a promising biotic pesticide produced by Chaetomium globosum. Recently, the regulatory function of Zn2Cys6 binuclear finger transcription factor CgcheR that lies within the ChA biosynthesis gene cluster has been confirmed. However, CgcheR was not merely a pathway specific regulator. In this study, we showed that the homologs gene of CgcheR (designated as Cgtf1) regulate ChA biosynthesis and sporulation in C. globosum NK102. More importantly, RNA-seq profiling demonstrated that 1,388 genes were significant differentially expressed as Cgtf1 deleted. Among them, a putative C2H2 transcription factor, named Cgtf6, showed the highest gene expression variation in zinc-binding proteins encoding genes as Cgtf1 deleted. qRT-PCR analysis confirmed that expression of Cgtf6 was significantly reduced in CgTF1 null mutants. Whereas, deletion of Cgtf6 resulted in the transcriptional activation and consequent increase in the expression of ChA biosynthesis gene cluster and ChA production in C. globosum. These data suggested that CgTF6 probably acted as an end product feedback effector, and interacted with CgTF1 to maintain a tolerable concentration of ChA for cell survival.

A Fungal Diterpene Synthase Is Responsible for Sterol Biosynthesis for Growth.

A conserved open reading frame, dps, is described in Pestalotiopsis microspora, sharing a remarkable similarity with fungal diterpene synthases whose function is less studied. Loss-of-function approach manifested that dps was necessary for the growth and the development of the fungus. A deletion strain, dpsΔ, showed a fundamental retardation in growth, which could deliberately be restored by the addition of exogenous sterols to the media. Gas chromatography-mass spectrometry analysis confirmed the loss of the ability to produce certain sterols. Thus, the tolerance and the resistance of dpsΔ to several stress conditions were impaired. Secondary metabolites, such as the polyketide derivative dibenzodioxocinones, were significantly diminished. At the molecular level, the deletion of dps even affected the expression of genes in the mevalonate pathway. This report adds knowledge about fungal diterpene synthases in Pestalitiopsis microspora.

Surface-enhanced Raman scattering analysis of serum albumin via adsorption-exfoliation on hydroxyapatite nanoparticles for noninvasive cancers screening.

Combining serum albumin via adsorption-exfoliation on hydroxyapatite particles (HAp) with surface-enhanced Raman scattering (SERS), we developed a novel quantitative analysis of albumin method from blood serum for cancers screening applications. The quantitatively analysis obtained by our HAp method had a good linear relationship from 1 to 10 g/dL, and the lower limit of detection was less than the albumin prognostic factor for disease (3.5 g/dL). Serum albumin was adsorbed and exfoliated by HAp from serum samples of liver cancer patients, breast cancer patients and healthy volunteers and mixed with silver colloids to perform SERS spectral analysis. Based on the PLS-SVM algorithm, the diagnostic accuracies of liver cancer patients and breast cancer patients were 100% and 96.68%, respectively. Moreover, this algorithm successfully predicted the unidentified subjects with a diagnostic accuracy of 93.75%. This exploratory work demonstrated that HAp-adsorbed-exfoliated serum proteins combined with SERS spectroscopy has great potential for cancer screening.

A Gene Cluster for the Biosynthesis of Dibenzodioxocinons in the Endophyte Pestalotiopsis microspora, a Taxol Producer.

The fungal products dibenzodioxocinones promise a novel class of inhibitors against cholesterol ester transfer protein (CEPT). Knowledge as to their biosynthesis is scarce. In this report, we characterized four more dibenzodioxocinones, which along with a previously described member pestalotiollide B, delimit the dominant spectrum of secondary metabolites in P. microspora. Through mRNA-seq profiling in gα1Δ, a process that halts the production of the dibenzodioxocinones, a gene cluster harboring 21 genes including a polyketide synthase, designated as pks8, was defined. Disruption of genes in the cluster led to loss of the compounds, concluding the anticipated role in the biosynthesis of the chemicals. The biosynthetic route to dibenzodioxocinones was temporarily speculated. This study reveals the genetic basis underlying the biosynthesis of dibenzodioxocinone in fungi, and may facilitate the practice for yield improvement in the drug development arena.

Stable expression of chimeric anti-CD3 receptors on mammalian cells for stimulation of antitumor immunity.

Expression of CD80 or CD86 costimulatory molecules on tumor cells can produce rejection of immunogenic but not poorly immunogenic tumors. We have previously shown that anti-CD3 single-chain antibodies expressed on the surface of cells can directly activate T cells. We therefore investigated whether anti-CD3 "receptors" could enhance CD86-mediated rejection of poorly immunogenic tumors. Expression of anti-CD3 receptors on cells was increased by introduction of membrane-proximal "spacer" domains containing glycosylation sites between the single-chain antibody and the transmembrane domain of the chimeric receptors. Removal of glycosylation sites in the spacer reduced surface expression due to increased shedding of chimeric receptors from the cell surface. Induction of T-cell proliferation by anti-CD3 receptors did not correlate with the expression level of chimeric protein, but rather depended on the physical properties of the spacer. Anti-CD3 receptors effectively induced T-cell cytotoxicity, whereas coexpression with CD80 or CD86 was required for generating T-cell proliferation and IL-2 secretion. Although expression of CD86 did not significantly delay the growth of poorly immunogenic B16-F1 tumors, expression of anti-CD3 receptors with CD86 produced complete tumor rejections in 50% of mice and induced significant protection against wild-type B16-F1 tumor cells. Our results show that spacer domains can dramatically influence the surface expression and the biological activity of chimeric antibody receptors. The strong antitumor activity produced by anti-CD3 receptors and CD86 on tumor cells indicates that this strategy may be beneficial for the gene-mediated therapy of poorly immunogenic tumors.