Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

NCCN Guidelines® Insights: Ovarian Cancer, Version 3.2022.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the United States, with less than half of patients living >5 years following diagnosis. The NCCN Guidelines for Ovarian Cancer provide recommendations for the diagnosis, evaluation, treatment, and follow-up for patients with ovarian, fallopian tube, and primary peritoneal cancers. These NCCN Guidelines Insights summarize the panel discussion behind recent important updates to the guidelines, including revised guidance on alternative chemotherapy regimens for patients with advanced age and/or comorbidities, a new algorithm for recurrent low-grade serous carcinoma based on developing research and novel therapeutic agents, and updated language regarding tumor molecular analysis applications in ovarian cancer.

Fibrinogen aptamer functionalized gold-coated iron-oxide nanoparticles for targeted imaging of thrombi.

Targeting of molecular constituents of thrombi with aptamer functionalized core-shell nanoparticles (CSN) allowed for high resolution clot delineation in T2-weighted magnetic resonance imaging. Meanwhile, the gold-coating demonstrated sufficient contrast capabilities in computed tomography (1697 HU µM-1). This targeted CSN formulation could allow for precise imaging of blood clots at low nanomolar concentrations.

Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX.

Current classes of cancer therapeutics have negative side effects stemming from off-target cytotoxicity. One way to avoid this would be to use a drug delivery system decorated with targeting moieties, such as an aptamer, if a targeted aptamer is available. In this study, aptamers were selected against acute myeloid leukemia (AML) cells expressing the MLL-AF9 oncogene through systematic evolution of ligands by exponential enrichment (SELEX). Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and three candidate sequences were identified. These sequences consisted of two 23-base primer regions flanking a 30-base central domain. Binding studies were performed using flow cytometry, and the lead sequence had a binding constant of 37.5 + / - 2.5 nM to AML cells, while displaying no binding to fibroblast or umbilical cord blood cells at 200 nM. A truncation study of the lead sequence was done using nine shortened sequences, and showed the 5' primer was not important for binding. The lead sequence was tested against seven AML patient cultures, and five cultures showed binding at 200 nM. In summary, a DNA aptamer specific to AML cells was developed and characterized for future drug-aptamer conjugates.

Development and Evaluation of a Quantitative Fluorescent Lateral Flow Immunoassay for Cystatin-C, a Renal Dysfunction Biomarker.

The diagnosis, prognosis, and control of chronic kidney disease rely on an understanding of the glomerular filtration rate (GFR). The renal clearance of the cystatin-C is closely associated with the GFR. Cystatin-C is a more suitable GFR marker than the commonly used creatinine. General techniques for cystatin-C calculation, such as particle-enhanced turbidimetric and nephelometric assay, are time-consuming and tedious. Here, we propose a rapid, quantitative immunoassay for the detection of cystatin-C. A fluorescence-based lateral-flow kit was developed in a sandwich format by using a monoclonal antibody. A Linear calibration was obtained over the clinical diagnostic range of 0.023-32 µg/mL and the limit of detection (LOD) was 0.023 µg/mL and the limit of quantification (LOQ) was 0.029 µg/mL. Average recoveries from spiked urine samples ranged from 96-100% and the coefficient of variation was less than 4% for both intra and inter-day assays with excellent repeatability. With the comparison with an ELISA kit, the developed kit is highly sensitive, performs well over the detection range, provides repeatable results in a short time, and can easily be used at point-of-care (POC), making it an ideal candidate for rapid testing in early detection, community screening for renal function disorders.

Optimized experimental pre-treatment strategy for temporary inhibition of islet amyloid polypeptide aggregation.

Islet amyloid polypeptide (IAPP) is a neuroendocrine hormone from pancreatic ß-cells. Misfolded, aggregated IAPP is believed to be toxic to islet cells and amyloid deposits in the pancreas are pathological hallmarks of type 2 diabetes. Rapid fibrillization of this peptide makes it difficult to study in its soluble form, impeding a better understanding of its role. In this study, a variety of popular pretreatment methods were tested for their ability to delay aggregation of IAPP, including solutions of hexafluoroisopropanol, sodium hydroxide, hydrochloric acid, phosphate buffered saline, ammonium hydroxide, as well as tris buffer at different pH and containing either calcium (II), zinc (II), or iron (II). Aggregation was assessed using the thioflavin T fluorescence assay as well as by transmission electron microscopy. Tris buffer at pH 8.1 containing Zn(II) was found to have the best balance of temporary inhibition of aggregation and biological relevance.

Ovarian Cancer, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology.

Epithelial ovarian cancer is the leading cause of death from gynecologic cancer in the United States and is the country's fifth most common cause of cancer mortality in women. A major challenge in treating ovarian cancer is that most patients have advanced disease at initial diagnosis. These NCCN Guidelines discuss cancers originating in the ovary, fallopian tube, or peritoneum, as these are all managed in a similar manner. Most of the recommendations are based on data from patients with the most common subtypes─high-grade serous and grade 2/3 endometrioid. The NCCN Guidelines also include recommendations specifically for patients with less common ovarian cancers, which in the guidelines include the following: carcinosarcoma, clear cell carcinoma, mucinous carcinoma, low-grade serous, grade 1 endometrioid, borderline epithelial, malignant sex cord-stromal, and malignant germ cell tumors. This manuscript focuses on certain aspects of primary treatment, including primary surgery, adjuvant therapy, and maintenance therapy options (including PARP inhibitors) after completion of first-line chemotherapy.

HER2 breast cancer biomarker detection using a sandwich optical fiber assay.

Optical fiber-based surface plasmon resonance (OF-SPR) sensors have demonstrated high versatility and performances over the last years, which propelled the technique to the heart of numerous and original biosensing concepts. In this work, we contribute to this effort and present our recent findings about the detection of breast cancer HER2 biomarkers through OF-SPR optrodes. 1 cm-long sections of 400 µm core-diameter optical fibers were covered with a sputtered gold film, yielding enhanced sensitivity to surface refractive index changes. Studying the impacts of the gold film thickness on the plasmonic spectral response, we improved the quality and reproducibility of the sensors. These achievements were correlated in two ways, using both the central wavelengths of the plasmon resonance and its influence on the bulk refractive index sensitivity. Our dataset was fed by additional biosensing experiments with a direct and indirect approach, relying on aptamers and antibodies specifically implemented in a sandwich layout. HER2 biomarkers were specifically detected at 0.6 µg/mL (5.16 nM) in label-free while the amplification with HER2-antibodies provided a nearly hundredfold signal magnification, reaching 9.3 ng/mL (77.4 pM). We believe that these results harbinger the way for their further use in biomedical samples.

HER2 biosensing through SPR-envelope tracking in plasmonic optical fiber gratings.

In the biomedical detection context, plasmonic tilted fiber Bragg gratings (TFBGs) have been demonstrated to be a very accurate and sensitive sensing tool, especially well-adapted for biochemical detection. In this work, we have developed an aptasensor following a triple strategy to improve the overall sensing performances and robustness. Single polarization fiber (SPF) is used as biosensor substrate while the demodulation is based on tracking a peculiar feature of the lower envelope of the cladding mode resonances spectrum. This method is highly sensitive and yields wavelength shifts several tens of times higher than the ones reported so far based on the tracking of individual modes of the spectrum. An amplification of the response is further performed through a sandwich assay by the use of specific antibodies. These improvements have been achieved on a biosensor developed for the detection of the HER2 (Human Epidermal Growth Factor Receptor-2) protein, a relevant breast cancer biomarker. These advanced developments can be very interesting for point-of-care biomedical measurements in a convenient practical way.

Multimodal plasmonic optical fiber grating aptasensor.

Tilted fiber Bragg gratings (TFBGs) are now a well-established technology in the scientific literature, bringing numerous advantages, especially for biodetection. Significant sensitivity improvements are achieved by exciting plasmon waves on their metal-coated surface. Nowadays, a large part of advances in this topic relies on new strategies aimed at providing sensitivity enhancements. In this work, TFBGs are produced in both single-mode and multimode telecommunication-grade optical fibers, and their relative performances are evaluated for refractometry and biosensing purposes. TFBGs are biofunctionalized with aptamers oriented against HER2 (Human Epidermal Growth Factor Receptor-2), a relevant protein biomarker for breast cancer diagnosis. In vitro assays confirm that the sensing performances of TFBGs in multimode fiber are higher or identical to those of their counterparts in single-mode fiber, respectively, when bulk refractometry or surface biosensing is considered. These observations are confirmed by numerical simulations. TFBGs in multimode fiber bring valuable practical assets, featuring a reduced spectral bandwidth for improved multiplexing possibilities enabling the detection of several biomarkers.

Rapid Detection of Circulating Breast Cancer Cells Using a Multiresonant Optical Fiber Aptasensor with Plasmonic Amplification.

The detection of circulating tumor cells (CTCs), which are responsible for metastasis in several forms of cancer, represents an important goal in oncological diagnosis and treatment. These cells remain extremely challenging to detect, despite numerous previous studies, due to their low concentration (1-10 cells/mL of blood). In this work, an all-fiber plasmonic aptasensor featuring multiple narrowband resonances in the near-infrared wavelength range was developed to detect metastatic breast cancer cells. To this aim, specific aptamers against mammaglobin-A were selected and immobilized as receptors on the sensor surface. In vitro assays confirm that the label-free and real-time detection of cancer cells [limit of detection (LOD) of 49 cells/mL] occurs within 5 min, while the additional use of functionalized gold nanoparticles allows a 2-fold amplification of the biosensor response. Differential measurements on selected optical resonances were used to process the sensor response, and results were confirmed by microscopy. The detection of only 10 cancer cells/mL was achieved with relevant specificity against control cells and with quick response time.

An aptamer-based colorimetric lateral flow assay for the detection of human epidermal growth factor receptor 2 (HER2).

An aptamer-based colorimetric lateral flow assay was developed for the detection of human epidermal growth factor receptor 2 (HER2). In this study, two approaches were examined using HER2 binding aptamers and gold nanoparticles. The first method used was a solution-based adsorption-desorption colorimetric approach wherein aptamers were adsorbed onto the gold nanoparticle surface. Upon the addition of HER2, HER2 binds specifically with its aptamer, releasing the gold nanoparticles. Addition of NaCl then induces the formation of gold nanoparticle aggregates. This leads to a color change from red to blue and a detection limit of 10 nM was achieved. The second method used an adsorption-desorption colorimetric lateral flow assay approach wherein biotin-modified aptamers were adsorbed onto the gold nanoparticle surface in the absence of HER2. In the presence of HER2, HER2 specifically binds with its aptamer leading to release of the gold nanoparticles. These solutions were applied to the lateral flow assay format and a detection limit of 20 nM was achieved. Both colorimetric and lateral flow assays are inexpensive, simple, rapid to perform and produce results visible to the naked-eye.

Comparison of turn-on and ratiometric fluorescent G-quadruplex aptasensor approaches for the detection of ATP.

Two fluorescent aptasensor methods were developed for the detection of ATP in biochemical systems. The first method consisted of a label-free fluorescent "turn-on" approach using a guanine-rich ATP aptamer sequence and the DNA-binding agent berberine complex. In the presence of ATP, the ATP preferentially binds with its aptamer and conformationally changes into a G-quadruplex structure. The association of berberine with the G-quadruplex results in the enhancement of the fluorescence signal of the former. The detection limit of ATP was found to be 3.5 µM. Fluorescence, circular dichroism and melting temperature (Tm) experiments were carried out to confirm the binding specificity and structural changes. The second method employs the ratiometric fluorescent approach based on the Forster resonance energy transfer (FRET) for the detection of ATP using berberine along with a quencher (AuNRs, AgNPs) and a fluorophore (red quantum dots (RQDs), carbon dots (CDs)) labeled at 5' and 3' termini of the ATP-binding aptamer sequence. Upon addition of ATP and berberine, ATP specifically binds with its aptamer leading to the formation of G-quadruplex, and similarly, berberine also binds to the G-quadruplex. This leads to an enhancement of fluorescence of berberine while that of RQD and CDs were significantly quenched via FRET. The respective detection limits calculated were 3.6 µM and 3.8 µM, indicating these fluorescent aptasensor methods may be used for a wide variety of small molecules. Graphical abstract.

In vitro selections of mammaglobin A and mammaglobin B aptamers for the recognition of circulating breast tumor cells.

Mammaglobin B (MGB2) and mammaglobin A (MGB1) are proteins expressed in metastatic breast cancers. The early detection of circulating tumor cells (CTCs) in breast cancer patients is crucial to decrease mortality rate. Herein, novel aptamers were successfully selected and characterized against MGB2 and MGB1 proteins using a hybrid SELEX approach. The potential use of the selected aptamers in breast CTC detection was studied using spiked breast cancer cells in whole blood lysate. The results obtained from this study showed that the selected aptamers (MAMB1 and MAMA2) bind to their target breast cancer cell lines with high affinity (low nanomolar Kd values) and specificity. They also bind to their free recombinant target proteins and show minimal non-specific binding to normal and other cancer cell lines. Additionally, they were able to distinguish a low number of breast cancer cells spiked in whole blood lysate containing normal blood cells. The results obtained in this study indicate the great potential for the use of aptamers to detect MGB1 and MGB2 protein biomarkers, expressed on the surface of breast CTCs.

Aptamers: Promising Tools for the Detection of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are cells that shed from a primary tumor and freely circulate in the blood, retaining the ability to initiate metastasis and form a secondary tumor in distant organs in the body. CTCs reflect the molecular profile of the primary tumor, therefore studying CTCs can allow for an understanding of the mechanism of metastasis, and an opportunity to monitor the prognosis of cancer. Unfortunately, the detection of CTCs is a considerable challenge due to their low abundance in the bloodstream and the lack of consistent markers present to recognize these cells. The aim of this review is to summarize some of the aptamer-based affinity methods for the detection of CTCs. The basic biological concept of how metastasis occurs and the role of CTCs in this process are presented. Some methods of CTC detection employing antibodies or peptides are mentioned here for comparison. The review of present literature suggests that aptamers are emerging as competitive technology in the detection of CTCs, especially due to their unique properties, but there still remain several challenges to be met, including the need to improve the throughput and sensitivity of such methods.

Ovarian Cancer, Version 1.2016, NCCN Clinical Practice Guidelines in Oncology.

This selection from the NCCN Guidelines for Ovarian Cancer focuses on the less common ovarian histopathologies (LCOHs), because new algorithms were added for LCOHs and current algorithms were revised for the 2016 update. The new LCOHs algorithms include clear cell carcinomas, mucinous carcinomas, and grade 1 (low-grade) serous carcinomas/endometrioid epithelial carcinomas. The LCOHs also include carcinosarcomas (malignant mixed Müllerian tumors of the ovary), borderline epithelial tumors (also known as low malignant potential tumors), malignant sex cord-stromal tumors, and malignant germ cell tumors.

Aptamers as promising molecular recognition elements for diagnostics and therapeutics in the central nervous system.

Oligonucleotide aptamers are short, synthetic, single-stranded DNA or RNA able to recognize and bind to a multitude of targets ranging from small molecules to cells. Aptamers have emerged as valuable tools for fundamental research, clinical diagnosis, and therapy. Due to their small size, strong target affinity, lack of immunogenicity, and ease of chemical modification, aptamers are an attractive alternative to other molecular recognition elements, such as antibodies. Although it is a challenging environment, the central nervous system and related molecular targets present an exciting potential area for aptamer research. Aptamers hold promise for targeted drug delivery, diagnostics, and therapeutics. Here we review recent advances in aptamer research for neurotransmitter and neurotoxin targets, demyelinating disease and spinal cord injury, cerebrovascular disorders, pathologies related to protein aggregation (Alzheimer's, Parkinson's, and prions), brain cancer (glioblastomas and gliomas), and regulation of receptor function. Challenges and limitations posed by the blood brain barrier are described. Future perspectives for the application of aptamers to the central nervous system are also discussed.