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- Development of an immunosensor based on quantum dots with different emissions for the detection of cancer biomarkersPublication . Oliveira, Daniela; Piloto, Ana Margarida L.(Introduction) Cancer is a global health challenge, where early detection is crucial to increasing survival rates [1]. Non-invasive, rapid, and affordable diagnostic methods are urgently needed. Immunosensors emerge as promising alternatives to traditional methods such as ELISA, allowing point-of-care (PoC) analyses [2]. This study introduces a fluorescent probe using cadmium telluride quantum dots (CdTe@MPA QDs) to detect the pancreatic cancer biomarker CA 19-9. With the increasing CA 19-9 concentrations, the fluorescence intensity of green-, orange-, and red-emitting QDs@conjugates was reduced, creating a visible colour gradient under a 365 nm UV lamp. The method, suitable for serum analysis, is highly sensitive and selective, with a detection range of 0.31 to 501.9 U mL-1 and analyzed using ImageJ software. These immunosensors offer significant potential for cancer biomarker detection in clinical diagnostics.
- New Quantum-Dot-Based Fluorescent Immunosensor for Cancer Biomarker DetectionPublication . Sousa, Mariana P.; Piloto, Ana Margarida L.; Pereira, Ana Cláudia; Schmitt, Fernando; Fernandes, Ruben; Moreira, Felismina T. C.Cancer antigen 15-3 (CA 15-3) is a biomarker for breast cancer used to monitor response to treatments and disease recurrence. The present work demonstrates the preparation and application of a fluorescent biosensor for ultrasensitive detection of the cancer antigen CA 15-3 protein tumor marker using mercaptopropionic-acid-functionalized cadmium telluride (CdTe@MPA) quantum dots (QDs) conjugated with CA 15-3 antibodies. First, the QDs were synthesized by the hydrothermal route, resulting in spherical nanoparticles up to 3.50 nm in diameter. Subsequently, the QD conjugates were characterized by Fourier transform infrared spectroscopy (FTIR), UV absorption, and fluorescence. The interaction between the conjugates and the protein was studied by fluorescence spectroscopy in buffer and in 10-fold diluted commercial human serum. Calibration in spiked serum samples gave a detection limit of 0.027 U/mL, 1000-fold lower than the clinical limit for CA 15-3 (25 U/mL to 30 U/mL), indicating that this is an ultrasensitive technique. In addition, a rapid response was obtained within 10 min. The biosensor was selective in the presence of the interfering serum proteins BSA, CEA, and CA-125, with a maximum interference of 2% for BSA. The percent recovery was close to 100% with maximum relative standard deviation (RSD%) values of 1.56. Overall, the developed CA 15-3 biosensor provides a simple and sensitive method for ultrasensitive monitoring of breast cancer, as well as the ability to detect other molecules of interest in human serum matrices.
- Quantum dot-based optical sensor for MMP7 biomarker with molecularly imprinted polymersPublication . Oliveira, Pedro; Piloto, Ana Margarida L.MMP7, also known as matrix metalloproteinase 7, is a biomarker for oncogenic activity in a variety of tumour types. As part of the zinc-dependent endopeptidases group, it can break down practically all protein components found in the extracellular matrix1. CdTe MPA-Capped Quantum Dots (CdTe@MPA QDs) are luminescent nanoparticles with exceptional optical features as customized particle sizes and narrow emission spectra. They can be integrated into sensing systems for the sensitive and selective detection of metal ions, biomarkers, and antibiotics2. QDs can be functionalized by in situ polymerization, which combines monomers, crosslinkers, and proteins to create recognition cavities. Upon rebinding with the target biomarker, these cavities demonstrate biomimetic recognition sites complementary to the target biomolecule. The presence of the target leads to fluorescence quenching, of the functionalized QDs with MIPs, thus allowing for a simple and straightforward method for the selective quantification of the biomolecule.
- Paper-based approach for cancer biomarker MMP7 detection using quantum dots and molecularly imprinted polymersPublication . Oliveira, Pedro; Piloto, Ana Margarida L.(Introduction) Preliminary investigation using lab-on-paper Molecular Imprinted Polymers (MIPs) paired with Quantum Dots (QDs) to detect Matrix Metalloproteinase-7 (MMP7) using image processing to quantify fluorescence and area with a CMOS sensor reflex camera. MMP7 is critical in pancreatic tumor formation, which has the lowest survival rate among malignant malignancies. High levels of the MMP7 in blood have been associated to a poor prognosis and limited survival in patients. CdTe Quantum Dots (CdTe@MPA QDs) are luminescent nanoparticles with narrow emission spectra, enabling for exceptional sensitive detection of biomarkers.Combination of MIPs with CdTe MPA-capped provide selectivitive for biomiarker.
- Label-free quantum dot conjugates for human protein IL-2 based on molecularly imprinted polymersPublication . Piloto, Ana Margarida L.; Ribeiro, David S.M.; Rodrigues, S. Sofia M.; Santos, João L.M.; Ferreira Sales, Maria GoretiHerein, the development of a fluorescent-based sensor by combining quantum dots (QDs) with molecularly-imprinted technology (MIT), intensively optimized to generate exceptional operating features is presented. This sensor is designed to target human interleukin-2 (IL-2) in synthetic human serum. IL-2 is a regulatory protein released as a triggered response from the immune system towards an inflammation. For this purpose, cadmium telluride (CdTe) QDs are prepared with 3-mercaptopropionic acid (MPA) and modified afterwards to produce an IL-2 imprinted polymer with methacrylic acid and N,N´-methylenebis(acrylamide), upon removal of the template under optimized conditions. During IL-2 rebinding, the fluorescence intensity of CdTe@MPA QDs is quenched in a concentration dependent manner. Using surface imprinting technology, the optimal fluorescence signals yielded a linear response versus logarithm of IL-2 concentration from 35 fg/ml to 39 pg/ml, in a 1000-fold diluted synthetic human serum. The limit of detection obtained is 5.91 fg/ml, lying below the concentration levels of IL-2 with clinical interest for cancer diagnosis (9.4–19.2 pg/ml). Overall, the method presented herein is a demonstration that the combination of MIP and QDs for protein detection constitutes a powerful tool in clinical analysis, providing low cost, sensitive and quick responses. The same concept may be further extended to other proteins of interest.
- Ratiometric determination of CA19-9 using fluorescent carbon dots and yellow-emitting quantum dotsPublication . Rodrigues, E.; Piloto, A.M.L.(Introduction) Quantum dots (QDs) have high fluorescence, chemical stability and sensitivity and optical properties. Traditional QDs probes have a single peak, leading to some problems such as systemic errors due to detection conditions and fluorescence background and systemic instability. Ratiometric methods eliminate random changes in QDs fluorescence intensity and reduces optical fluctuations not caused by the target.
- Detection of CA19-9 using fluorescent polymer imprinted membranesPublication . Xu, Ana; Piloto, Ana Margarida L.(Introduction) The Carbohydrate antigen19-9 (CA19-9) is a biomarker widely used for the diagnosis and monitoring of various types of cancer, especially pancreatic cancer (PC). Molecularly Imprinted Polymers (MIPs) are synthetic materials with recognition sites molecularly designed to bind selectively to a target analyte, providing high specificity and sensitivity. In this work, the use of solid supports such as membranes, was used to imprint the MIPs, allowing the creation of robust and easy-to-manipulate biomimetic sensors for the precise and efficient detection of biomarkers, specifically CA19-9.
- Ratiometric optical visual immunosensor based on quantum dots for CA 19-9 protein detectionPublication . Oliveira, Daniela; Piloto, Ana Margarida L.Cancer is a global health challenge, where early detection is crucial to increasing survival rates. Non-invasive, rapid, and affordable diagnostic methods are urgently needed. Immunosensors emerge as promising alternatives to traditional methods such as ELISA, allowing point-of-care (PoC) analyses. This study aims to develop a highly sensitive and selective optical immunosensor for detecting CA 19-9. The results demonstrate successful detection of CA 19-9 over a wide concentration range from 1.77 to 501.87 U mL-1 in 1000-fold diluted human serum in just 10 minutes, with high precision. This device offers rapid, sensitive, and affordable detection, making it suitable for clinical PoC applications.
- Molecularly imprinted polymer for Mucin 4 (MUC4) biomarkerPublication . Piloto, A.M.L.; Cerqueira, N.; Sales, Goreti(Introduction) Molecularly imprinted polymers (MIPs) were synthesized around quantum dots (QDs) to detect the protein biomarker MUC4, which is associated with pancreatic cancer (Kauretal.,2022; Mehrotraetal.,2024). In this approach, MIPs serve as biorecognition elements and are conjugated with cadmium telluride QDs to form the sensing system. The fluorescence of these imprinted polymers is quenched as MUC4 concentrations increase, demonstrating a linear response within the range of 0.12 ng/mL to 16.9 ng/mL in human serum. This range is below the pancreatic cancer diagnostic cut-off values (1.2 to 1.3 ng/mL). The MIPs exhibit good stability and selectivity for MUC4 compared to non-imprinted controls. Additionally, theoretical studies provide valuable insights for the prediction of the functional structure of MIPs, enhancing its selectivity for the target, while reducing time and costs associated with laborious benchtop assays, which may provide an effective tool for tailoring MIPs to other biomarkers for clinical diagnosis.
- Sensitive ratiometric imprinted hydrogel for the detection of matrix metalloproteinase 7 (MMP7) biomarkerPublication . Piloto, Ana Margarida L.; Ribeiro, David S.M.; Santos, João L.M.; Sales, GoretiA dual-emissive fluorescent probe has been developed for the sensitive and selective detection of matrix metalloproteinase 7 (MMP7), a protein biomarker associated with pancreatic cancer1,2. The resultant materials were designated as imprinted ratiometric hydrogels (imprinted rHGs). The fluorescence quenching of the imprinted rHGs occurred with increasing concentrations of MMP7, showing linearity in the range [1.49 x 10-11 – 1.92 x 10-9] g/mL in 1000-fold diluted human serum in PBS 10mM pH 5.8. Overall, the imprinted rHGs developed in this work presented increased selectivity for the MMP7 protein over raw emitting QDs nanoparticles and higher sensitivity comparatively to the non-imprinted rHGs.