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- Development of a 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 was developed for the sensitive and selective detection of matrix metalloproteinase 7 (MMP7), a protein biomarker associated with pancreatic cancer. The ratiometric probe consisting of blue emitting carbon dots (CDs) and molecularly imprinted polymers (MIPs) previously assembled around red emitting quantum dots (QDs) was successfully combined for the detection of MMP7 protein. The concept is to use MIPs that function as the biorecognition elements, conjugated to cadmium telluride QDs, as the sensing system. The fluorescence intensity of red QDs is quenched with increasing concentrations of the analyte, acting as sensitive probes, while the fluorescence intensity of blue emitting CDs remains constant, acting as internal controls. The resultant fluorescence color changed from red to blue as a function of the MMP7 concentration, under a 365 nm UV lamp. The imprinted material MIP@QDs were successfully incorporated within a cellulose hydrogel containing CDs as reference probes. 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 × 10–11 – 1.92 × 10–9] g/mL, in 1000-fold diluted human serum. The imprinted rHGs showed an imprinting factor of 1.83 and a limit of detection of 4.11 × 10–12 g/mL. Overall, the imprinted rHGs developed in this work presented increased selectivity for the MMP7 protein over red emitting QDs nanoparticles and higher sensitivity comparatively to the non-imprinted rHGs.
- 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.
- 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.
- 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.
- Optical detection of CA19-9 protein using yellow quantum dots with molecularly imprinted polymersPublication . Xu, Ana; Piloto, Ana Margarida L.(Introduction) Biosensors are crucial for rapid and accurate diagnoses, vital for effective treatment strategies. ❖ Traditional immunoassays have susceptibility to instability and compromise the accuracy of the results. Molecularly imprinted polymers (MIP) are emerging as biomimetic sensors that offer stability, selectivity, and resistance to environmental variations. Quantum dots (QDs) have versatile optical detection probes, raising the sensitivity of detection methods. This work aims to create a biomimetic sensor for the detection of the biomarker CA19-9, associated with pancreatic cancer.
- Cellulose‑based hydrogel on quantum dots with molecularly imprinted polymers for the detection of CA19‑9 protein cancer biomarkerPublication . Piloto, Ana Margarida L.; Ribeiro, David S.M.; Rodrigues, S. Sofia M.; Santos, João L.M.; Sampaio, Paula; Sales, Maria Goreti FerreiraMolecularly imprinted polymers (MIPs), were successfully assembled around quantum dots (QDs), for the detection of the carbohydrate antigen CA19-9, a biomarker associated with pancreatic cancer (PC). The imprinted materials MIP@QDs were incorporated within cellulose hydrogels and designated as imprinted hydrogels (iHGs). The concept is to use MIPs which function as the biorecognition elements, conjugated to QDs as the sensing system. The fluorescence quenching of the iHGs occurred with increasing concentrations of CA19-9, showing linearity in the range 2.76 × 10−2 – 5.23 × 102 U/ml, in a 1000-fold diluted human serum. The iHGs show a linear response below the cutoff values for pancreatic cancer diagnosis (< 23 U/ml), a limit of detection of 1.58 × 10−3 U/ml and an imprinting factor (IF) of 1.76. Moreover, they exhibit stability and selectivity towards CA19-9, when compared with the non-imprinted controls, here designated as non-imprinted hydrogels (non-iHGs). In conclusion, this work demonstrates that the conjugation of MIPs to QDs increases the sensitivity and specificity of the developed sensing system for optical detection methods within clinical significance ranges. This fact shows potential for the iHGs to be applied as a sensitive, low-cost format for point-of-care tests (PoCTs).