A novel aptamer-based histochemistry assay for specific diagnosis of clinical breast cancer tissues

Pathological detection using immunohistochemistry (IHC) has become an indispensable process in the diagnosis confirmation of various cancers. However, the production of monoclonal antibodies is always very complex, expensive and time-consuming, and the batch differences are significant due to the corporeity and health statuses of animals may be different. In this work, an aptamer-based histochemistry (aptahistochemistry) assay was developed using a DNA aptamer for specific diagnosis of clinical breast cancer tissue sections. This aptahistochemistry assay can specifically distinguish Luminal A breast cancer molecular subtype from Luminal B (HER2+), HER2-enriched, and triple-negative breast cancer molecular subtypes, as well as para-carcinoma tissue, mastitis tissue and normal breast tissue. The accuracy of this aptahistochemistry assay for the diagnosis of Luminal A breast cancer was as high as 80%, which showed a great potential for clinical pathological diagnosis applications.     

A novel aptamer functionalized CuInS2 quantum dots probe for daunorubicin sensing and near infrared imaging of prostate cancer cells

In this paper, a novel daunorubicin (DNR)-loaded MUC1 aptamer-near infrared (NIR) CuInS₂ quantum dot (DNR–MUC1–QDs) conjugates were developed, which can be used as a targeted cancer imaging and sensing system. After the NIR CuInS₂ QDs conjugated with the MUC1 aptamer–(CGA)₇, DNR can intercalate into the double-stranded CG sequence of the MUC1–QDs. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of DNR on these conjugates. DNR–MUC1–QDs can be used to target prostate cancer cells. We evaluated the capacity of MUC1–CuInS₂ QDs for delivering DNR to cancer cells in vitro, and its binding affinity to MUC1-positive and MUC1-negative cells. This novel aptamer functionalized QDs bio-nano-system can not only deliver DNR to the targeted prostate cancer cells, but also can sense DNR by the change of photoluminescence intensity of CuInS₂ QDs, which concurrently images the cancer cells. The quenched fluorescence intensity of MUC1–QDs was proportional to the concentration of DNR in the concentration ranges of 33–88 nmol L⁻¹. The detection limit (LOD) for DNR was 19 nmol L⁻¹. We demonstrate the specificity and sensitivity of this DNR–MUC1–QDs probe as a cancer cell imaging, therapy and sensing system in vitro.     

Electrogenerated chemiluminescence biosensing for the detection of prostate PC-3 cancer cells incorporating antibody as capture probe and ruthenium complex-labelled wheat germ agglutinin as signal probe

A highly selective and sensitive electrogenerated chemiluminescence (ECL) biosensor for the detection of prostate PC-3 cancer cells was designed using a prostate specific antibody as a capture probe and ruthenium complex-labelled wheat germ agglutinin as a signal probe. The ECL biosensor was fabricated by covalently immobilising the capture probe on a graphene oxide-coated glassy carbon electrode. Target PC-3 cells were selectively captured on the surface of the biosensor, and then, the signal probe was bound with the captured PC-3 cells to form a sandwich. In the presence of tripropylamine, the ECL intensity of the sandwich biosensor was logarithmically directly proportion to the concentration of PC-3 cells over a range from 7.0 × 10² to 3.0 × 10⁴ cells mL⁻¹, with a detection limit of 2.6 × 10² cells mL⁻¹. The ECL biosensor was also applied to detect prostate specific antigen with a detection limit of 0.1 ng mL⁻¹. The high selectivity of the biosensor was demonstrated in comparison with that of a lectin-based biosensor. The strategy developed in this study may be a promising approach and could be extended to the design of ECL biosensors for highly sensitive and selective detection of other cancer-related cells or cancer biomarkers using different probes.     

A novel sensing platform using aptamer and RNA polymerase-based amplification for detection of cancer cells

Cancer is one of the most serious and lethal diseases around the world. Its early detection has become a challenging goal. To address this challenge, we developed a novel sensing platform using aptamer and RNA polymerase-based amplification for the detection of cancer cells. The assay uses the aptamer as a capture probe to recognize and bind the tumor marker on the surface of the cancer cells, forming an aptamer-based sandwich structure for collection of the cells in the microplate wells, and uses SYBR Green II dye as a tracer to produce strong fluorescence signal. The tumor marker interacts first with the recognition probes which were composed of the aptamer and single-stranded T7 RNA polymerase promoter. Then, the recognition probe hybridized with template probes to form a double-stranded T7 RNA polymerase promoter. This dsDNA region is extensively transcribed by T7 RNA polymerase to produce large amounts of RNAs, which are easily monitored using the SYBR Green II dye and a standard fluorometer, resulting in the amplification of the fluorescence signal. Using MCF-7 breast cancer cell as the model cell, the present sensing platform showed a linear range from 5.0 × 10² to 5.0 × 10⁶ cells mL⁻¹ with a detection limit of 5.0 × 10² cells mL⁻¹. This work suggested a strategy to use RNA signal amplification combining aptamer recognition to develop a highly sensitive and selective method for cancer cells detection.     

Selection of a DNA aptamer for the development of fluorescent aptasensor for carbaryl detection

An improved ssDNA library immobilized systematic evolution of ligands by enrichment(SELEX) was applied to select aptamers against carbaryl.After nine selection rounds,a highly enriched ssDNA pool was obtained.The Apta3 was demonstrated as the optimal aptame r.In order to facilitate the modification of aptamer,the Apta3 was further truncated with the dissociation constant(K_d) of 0.3 64 ± 0.055 μmol/L and a fluorescent aptasensor was developed.The linear range for carbaryl was from 100 nmol/L to1500 nmol/L,with the limit of detection was as low as 15.23 nmol/L.Besides,the biosensor was validated for the carbaryl spiked real samples,and the recoveries were between 97.7% and 107.3%.     

A novel bidentate ligand containing oxime,hydrazone and indole moieties and its BF2+ bridged transition metal complexes and their efficiency against prostate and breast cancer cells

In this study, a novel bidentate ligand containing oxime, hydrazone, and indole moieties and its BF₂⁺-bridged transition metal complexes [Ni(II), Cu(II), and Co(II)] were synthesized and their cytotoxic activities against prostate and breast cancer cells were investigated. The vic-dioxime ligand bearing indole–hydrazone side groups was synthesized by reacting antiglyoximehydrazine (GH₂) with 3-methoxy indole. The ligand forms mononuclear complexes with a metal-to-ligand ratio of 1:2 with M = Co(II)(H₂O)₂, Ni(II), and Cu(II). These metal complexes were then reacted with BF₃(C₂H₅)₂O to obtain BF₂⁺-bridged transition metal complexes. The Co(II) complex of the ligand is proposed to be octahedral with water molecules as axial ligands, whereas the Ni(II) and Cu(II) complexes are proposed to be square planar. Spectral studies showed that the ligand bonded to the metal ion in a neutral bidentate fashion through the azomethine nitrogen atom and the imine oxime group. Structural assignments are supported by a combination of ¹H nuclear magnetic resonance (NMR), ¹³C NMR, Fourier-transform infrared, LC/MS, elemental analyses, and magnetic susceptibility testing. For determining the cytotoxic effects of the novel anticancer products, cancer cells were cultured. The antiproliferative effects were determined using the MCF-7 breast cancer and PC-3 prostate cancer cell lines. The antiproliferative effects of the products were analyzed and their apoptotic or necrotic effects were determined with the Hoechst/propidium iodide double staining method in both cancer cell lines. Paclitaxel was used as the positive control (1 μm ). The results indicated that the newly synthesized compounds are effective on both cell lines between concentrations of 5 and 40 μm and show their effects by apoptotic mechanisms. Besides, these products were found to be more effective on the MCF-7 cell line. The cytotoxic efficiency of the newly synthesized products was more than that of paclitaxel (depending on concentration), which is a chemotherapeutic agent used in cancer therapy.     

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

胃腺癌核酸适配体的筛选与特异性研究

核酸适配体可作为分子探针应用于胃腺癌的早期诊断和治疗,具有广泛应用前景。本实验利用Serum-SELEX（Ser-SELEX）技术筛选胃腺癌核酸适配体。通过对候选适配体二级结构分析,其二级结构多为茎环结构。圆二色谱分析显示,7条候选核酸适配体呈右手螺旋特征。通过荧光定量核酸扩增检测系统（q-PCR）检测了候选核酸适配体对胃腺癌靶标的亲和力,表明候选核酸适配体浓度梯度与Ct值均呈正相关,亲和力常数为纳摩尔级别。利用q-PCR法、量子点法验证了候选核酸适配体识别靶标的特异性,结果均显示Apt-101对靶标亲和力更高,特异性更强,Apt-101的平衡解离常数（K_d值）为6.444±1.128nmol/L,特异性检测阳性率大于70%。     

Organotin(IV) 4-nitrophenylethanoates: Synthesis, structural characteristics and intercalative mode of interaction with DNA

Four new organotin(IV) carboxylates, [Bu₂SnL₂] (1), [Et₂SnL₂] (2), [Bu₃SnL]_n (3), [Me₃SnL]_n (4), where L = 4-nitrophenylethanoates, were synthesized and characterized by elemental analysis, FT-IR and multinuclear NMR (¹H and ¹³C). Spectroscopic results authenticated the coordination of ligand to the organotin moiety via COO group while X-ray single crystal analysis revealed bidentate chelating mode of coordination of ligand in complex 2 and a bridging behavior in complexes 3 and 4. Cyclic voltammetric (CV) technique was used to evaluate the electrochemical, kinetic and thermodynamic parameters of complexes 1-4, interacting with DNA. The linearity of the plots between the peak current (I) and the square root of the scan rate (ν^1/2) indicated the electrochemical processes to be diffusion controlled. The diffusion coefficients of the free (D_f) and DNA bound forms (D_b), standard rate constants (ks) and charge transfer coefficients (α) were determined by the application of Randle–Sevcik, Nicholson and Kochi equations. Furthermore, the binding constants evaluated from voltammetric data revealed the following increasing order of binding strength: 2 < 1 < 4 < 3. For 1 and 2, the activity against prostate cancer cell lines (PC-3) was found consistent with the order obtained from voltammetric behavior.     

Biomolecular profiling of metastatic prostate cancer cells in bone marrow tissue using FTIR microspectroscopy: a pilot study

Prostate cancer (CaP) cells preferentially metastasise to the bone marrow, a microenvironment that plays a substantial role in the sustenance and progression of the CaP tumour. Here we use a combination of FTIR microspectroscopy and histological stains to increase molecular specificity and probe the biochemistry of metastatic CaP cells in bone marrow tissue derived from a limited source of paraffin-embedded biopsies of different patients. This provides distinction between the following dominant metabolic processes driving the proliferation of the metastatic cells in each of these biopsies: glycerophospholipid synthesis from triacylglyceride, available from surrounding adipocytes, in specimen 1, through significantly high (p ≤ 0.05) carbohydrate (8.23 ± 1.44 cm⁻¹), phosphate (6.13 ± 1.5 cm⁻¹) and lipid hydrocarbon (24.14 ± 5.9 cm⁻¹) signals compared with the organ-confined CaP control (OC CaP), together with vacuolation of cell cytoplasm; glycolipid synthesis in specimen 2, through significantly high (p ≤ 0.05) carbohydrate (5.51 ± 0.04 cm⁻¹) and high lipid hydrocarbon (17.91 ± 2.3 cm⁻¹) compared with OC CaP, together with positive diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells; glycolysis in specimen 3, though significantly high (p ≤ 0.05) carbohydrate (8.86 ± 1.78 cm⁻¹) and significantly lower (p ≤ 0.05) lipid hydrocarbon (11.67 ± 0.4 cm⁻¹) than OC CaP, together with negative diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells. Detailed understanding of the biochemistry underpinning the proliferation of tumour cells at metastatic sites may help towards refining chemotherapeutic treatment.     

Link test--A statistical method for finding prostate cancer biomarkers

We present a new method, link-test, to select prostate cancer biomarkers from SELDI mass spectrometry and microarray data sets. Biomarkers selected by link-test are supported by data sets from both mRNA and protein levels, and therefore results in improved robustness. Link-test determines the level of significance of the association between a microarray marker and a specific mass spectrum marker by constructing background mass spectra distributions estimated by all human protein sequences in the SWISS-PROT database. The data set consist of both microarray and mass spectrometry data from prostate cancer patients and healthy controls. A list of statistically justified prostate cancer biomarkers is reported by link-test. Cross-validation results show high prediction accuracy using the identified biomarker panel. We also employ a text-mining approach with OMIM database to validate the cancer biomarkers. The study with link-test represents one of the first cross-platform studies of cancer biomarkers.     

Dual recognition strategy for ultra-sensitive fluorescent detection of Hg2+ at femto-molar level based on aptamer functionalized sulfur quantum dots

In the present study, a dual recognition strategy for ultrasensitive detection of Hg²⁺ was successfully developed for the first time based on aptamer functionalized sulfur quantum dots (Apt-SQDs). The developed Apt-SQDs not only retained the good fluorescence properties of quantum dots but also overcame the problem of poor selectivity of SQDs for heavy metal ions. This system used the dual recognition strategy, including the combination of S_x^2? and Hg²⁺ and T-Hg²⁺-T structures to excellently identify and capture Hg²⁺, and an ultrahigh sensitivity fluorescent aptasensor was fabricated. The fluorescent aptasensor had a good response to Hg²⁺ at concentrations ranging of 10^?15 to 10^?7 M with an ultralow limit of detection of 0.3 fM, and the response to other metal ions was far less than that to Hg²⁺. It was successfully applied to detect Hg²⁺ in nearby environmental water samples (tap water, lake water and river water) with a good recovery rate. Moreover, portable test papers that would be useful for Hg²⁺ monitoring in environmental water were designed. The dual recognition strategy not only achieves ultrasensitive fluorescent detection of Hg²⁺ but also provides a new insight into the further expansion of the application of SQDs.     

亲水作用色谱法测定组织中全基因组DNA甲基化水平

建立了亲水作用色谱(HILIC)测定组织中全基因组DNA甲基化水平的方法。采用苯酚-氯仿提取组织中的DNA,提取的DNA用88%甲酸在140 ℃下裂解,经N2吹干后,加乙腈-水(9:1, v/v)溶解,用Waters BEH HILIC柱进行分离,在277 nm波长下检测胞嘧啶(Cyt)及5-甲基胞嘧啶(5-mCyt)含量。结果表明,以乙腈-10 mmol/L甲酸铵溶液(94:6, v/v)为流动相,流速为0.5 mL/min, Cyt与5-mCyt分离较好,保留时间分别为2.6与3.1 min。胞嘧啶的线性范围为1～900 μmol/L,相关系数为0.9999; 5-甲基胞嘧啶的线性范围为1～64 μmol/L,相关系数为0.9998。胞嘧啶和5-甲基胞嘧啶的检出限为54 nmol/L(柱中为0.54 pmol),定量限为250 nmol/L(柱中为2.5 pmol);在5～900 μmol/L的添加水平下,胞嘧啶和5-甲基胞嘧啶的平均加标回收率为94.7%～100.5%,相对标准偏差小于1.48%。用该方法检测了结肠癌组织中DNA甲基化水平,结果显示该癌组织中全基因组的DNA甲基化均值为4.0%。该方法快速、简单,稳定性好,灵敏度较高,能满足全基因组DNA甲基化的检测要求。     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

Aptamer based fluorescent probe for serum HER2-ECD detection: The clinical utility in breast cancer

HB5 aptamer-based probe has been developed for serum HER2-ECD test in auxiliary clinical diagnosis and treatment for HER2-positive breast cancer patients.     

Horseradish peroxidase and aptamer dual-functionalized nanoprobe for the amplification detection of alpha-methylacyl-CoA racemase

Alpha-methylacyl-CoA racemase (AMACR) is over-expressed in many cancer types and can serve as a novel diagnostic biomarker. Development of convenient and sensitive detection methods of AMACR is of particular importance for cancer diagnosis. Aptamers are a type of recognition elements, which possess many advantages over antibody, making them suitable for applications in biosensing and biotechnology. In this work, we use the efficient surface modification of gold nanoparticles (AuNPs) to prepare the horseradish peroxidase (HRP) and aptamer dual-functionalized nanoprobe. The immobilization of HRP and thiol-terminated aptamer on the surface of AuNPs can be achieved through electrostatic interaction and the formation of Au–S bond, respectively. This nanoprobe, which is used as discriminating and catalytic probe, can be combined with enzyme immunoassay method to increase the detection sensitivity of AMACR. The detection limit can reach as low as 4.6 pg mL⁻¹ due to the dual signal amplification from enzymatic cycling and the high loading of enzymes on AuNPs. This sensitivity is about three orders of magnitude higher than that of AMACR aptamer based fluorescence method, which is also comparable to or one order of magnitude higher than that of ELISA. Furthermore, this method is more simple and effective, which not only avoids the conjugation between recognition element and the catalytic enzyme, but also achieves greater signal amplification. This assay could be used as a sensitive and selective platform for the detection of target protein.     

In vitro antitumor activity,ADME-Tox and 3D-QSAR of synthesized and selected natural styryl lactones

Prostate cancer is a common cause of death in men and a novel treating methods should be developed. In order to find a new drug for prostate cancer, a series of novel conformationally constrained analogues of (+)-goniofufurone and 7-epi-(+)-goniofufurone, as well as the newly synthesized styryl lactones containing the cinnamic acid ester groups were evaluated for in vitro cytotoxicity against prostate cancer cell (PC-3). Furthermore, prediction of physicochemical characteristics and drugability as well as in silico ADME-Tox tests of investigated compounds were performed. The 3D-QSAR model was established using the comparative molecular field analysis method. According to obtained results, the tricyclic compounds 9 and 10 had the highest potency with IC₅₀ < 20 μM. Evaluation of structural features through 3D-QSAR model identified steric field feature on the cinnamic acid ester groups at C-7 as a crucial for the cytotoxic activity. This research suggests that most of the analysed compounds have desirable properties for drug candidates and high potential in drug development, which recommend them for further research in treatment of prostate cancer. Furthermore, obtained 3D-QSAR model is able to successfully identify styryl lactones that have significant cytotoxic activity and provide information for screening and design of novel inhibitors against PC-3 cell line that could be used as drugs in treatment of the prostate cancer.     

Designing DNA cage-based immuno-fluorescence strategy for rapid diagnosis of clinical cervical cancer tissues

Exploiting a tissue diagnosis method to abstain the involuted operating and consume valuable reagents while realizing high-speed and inexpensive pathological grading technology to supply a better scheme for cancer therapy is a significant method of cancers detection. A promising immuno-fluorescence strategy was rationally designed and synthesized by loading ruthenium complex into cervical cancer-targeted DNA-cage, which was well used to realize high-speed and inexpensive diagnosis of clinical ce...     

Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization

A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamer–cell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinity interaction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specific binding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore no aptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result the solution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probes and aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purple solution. UV–vis spectrometry demonstrated that this hybridization-based method exhibited selective colorimetric responses to the presence or absence of target cells, which is detectable with naked eye. The linear response for MCF-7 cells in a concentration range from 10 to 10⁵ cells was obtained with a detection limit of 10 cells. The proposed method could be extended to detect other cells and showed potential applications in cancer cell detection and early cancer diagnosis.