Líneas de Investigación
TAILORING SELEX PROCEDURE TO SELECT NOVEL AND HIGHLY DIRECTED APTAMERS
We have selected the first aptamer for gluten (patented), the group of proteins that triggers the celiac disease, an elusive but desired target due to their poor solubility in water and apolar character. By using a subrogate, the most immunogenic peptide of gluten called 33-mer and magnetic particle technology we obtained several aptamers that binds not only the peptide but also the whole protein where the peptide is in its natural form.
We have use them in competitive formats to detect not only intact gluten but also hydrolyzed one (beers, infant formulas, etc) with lower limits of detection than the commercial immunoassays. We designed assays in easy-to-use format (magnetic particles) or disposable biosensors.
To detect gluten it is compulsory an extraction in ethanolic mixtures and further dilution to make compatible with the receptor (antibody or aptamer). Recently, we have first demonstrated the DES-SELEX, SELEX performed in deep eutectic solvents, green solvents with high solubility power that eliminates the need for sample dilution because the gluten is extracted in DES and directly interacts with DES-derived aptamers. This opens the way to obtain aptamers for poor soluble compounds and pushed down the limit of detection of gluten.
Aptamers for tumor biomarkers
Aberrant glycosylation is a hallmark of cancer. Virtually all tumor cells carry abnormal number/pattern of glycans at their membrane proteins. Serum reflect the pathological stage because contains cellular components from both normal and tumor cells. Most tumor biomarkers are glycoproteins but they still have concerns about its sensitivity (no false negative) and specificity (no false positive). It is believed that quantitation of aberrant glycasylation instead of its total content could improve biomarker usefulness in cancer diagnostics, prognostic and prediction but it compulsory requires novel receptor that recognize these features. Carbohydrates are low immunogenic so antibodies are difficult to raise. In addition the receptors elicited usually present low affinity. Aptamer, however, can be obtained for non-immunogenic compounds, are more easily directed toward a specific epitope and can be tailored to be stronger binders.
We have recently demonstrated the feasibilty of selecting aptamers for the glycan moiety of a glycoprotein such as PSA (the only biomarker approved for diagnose a tumor, the prostate cancer).
LIQUID BIOPSY: DETECTION OF CANCER BIOMARKERS
Cancer diagnosis ultimately relies on confirmatory biopsies, which are so invasive that cannot be repeated to monitor the progression/regression of the disease. This is the reason behind the concept of liquid biopsy, the development of analytical tools to detect biomolecules such as proteins, circulating DNA, circulating tumor cells, microRNA, lncRNA, exosomes, microvesicules in biological fluids that serve as disease biomarkers to eliminate invasive biopsies.
We have developed a sandwich aptasensor for PSA using our best aptamer recognizing the glycan region and a previously reported one recognizing the protein moiety that shows promising value in better discriminating patients with benign prostate disease from those with cancer than total PSA.
b) Circulating nucleic acids
Non-coding nucleic acids were regarded until the new century as "dark/trash matter" but the advances in massive sequencing and analytical methods of detection have revealed significant roles in the cellular metabolism. Both short (microRNA) and long non-coding RNAs are being associated to tumorigenesis and their level of expression of some of them is altered in cancer patients opening the way to become a tumor biomarker. Detection of both types of RNA has its own difficulties beyond those we have already addressed for detection of specific DNA sequences. They are frequently in extremely low levels (fM or lower).
ISOTHERMAL NUCLEIC ACID AMPLIFICATION TECHNIQUES
PCR is the gold standard technique for nucleic acid amplification but requires thermal cycling that makes more difficult miniaturization and integration in simple disposable platforms. Isothermal strategies mimicks in-vivo replication by replacing temperature changes for appropriate enzymes.
We have demonstrated that helicase dependent amplification (HDA) provides similar limits of detection than PCR. As a proof of concept, Mycobacterium tuberculosis was detected in biological specimens using HDA combined with a magnetoassay with electrochemical detection.
Amplification of aptamers after target recognition opens new avenues for signal amplification that are forbidden to antibodies. We have explored the amplification power of Rolling Circle Amplification (RCA) We have designed a shorter (just 15 min) and cheaper (four to ten times lower enzyme concentrations) RCA protocol and quantified the improvement of aptamer-biomarker recognition in up to three orders of magnitude.