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CRISPR-based diagnostics enable specific sensing of DNA and RNA biomarkers associated with human diseases. This is achieved through the binding of guide RNAs to a complementary sequence which activates Cas enzymes to cleave reporter molecules. Currently, most CRISPRbased diagnostics rely on target preamplification to reach sufficient sensitivity for clinical applications. This limits quantification capability and adds complexity to the reaction chemistry. Here, we show the combination of a CRISPR/Cas-based reaction with a Nanozyme-Linked ImmunoSorbent Assay which allows for the quantitative and colorimetric readout of Cas13- mediated RNA detection through catalytic metallic nanoparticles at room temperature (CrisprZyme). We demonstrate CrisprZyme is easily adaptable to a lateral-flow-based readout and different Cas enzymes, and enables the sensing of non-coding RNAs including microRNAs, long non-coding RNAs and circular RNAs. We utilise this platform to identify patients with acute myocardial infarction and to monitor cellular differentiation in vitro and in tissue biopsies from prostate cancer patients. We anticipate that CrisprZyme has significant potential as a universally applicable signal catalyst for CRISPR-based diagnostics which will expand the spectrum of targets for preamplification-free, quantitative detection.

Liposome-based drug delivery systems have allowed for better drug tolerability and longer circulation times but are often optimized for a single agent due to the inherent difficulty of co-encapsulating two drugs with differing chemical profiles. Here, we design and test a prodrug based on a ribosylated nucleoside form of 5-fluorouracil, 5-fluorouridine (5FUR), with the final purpose of co-encapsulation with doxorubicin (DOX) in liposomes. To improve the loading of 5FUR, we developed two 5FUR prodrugs that involved the conjugation of either one or three moieties of tryptophan (W) known respectively as, 5FUR&minus, W and 5FUR&minus, W3. 5FUR&minus, W demonstrated greater chemical stability than 5FUR&minus, W3 and allowed for improved loading with fewer possible byproducts from tryptophan hydrolysis. Varied drug ratios of 5FUR&minus, W: DOX were encapsulated for in vivo testing in the highly aggressive 4T1 murine breast cancer model. A liposomal molar ratio of 2.5 5FUR&minus, W: DOX achieved a 62.6% reduction in tumor size compared to the untreated control group and a 33% reduction compared to clinical doxorubicin liposomes in a proof-of-concept study to demonstrate the viability of the co-encapsulated liposomes. We believe that the new prodrug 5FUR&minus, W demonstrates a prodrug design with clinical translatability by reducing the number of byproducts produced by the hydrolysis of tryptophan, while also allowing for loading flexibility.

Personalized medicine is pushing forward new diagnostic techniques to aid in controlling drug therapeutic levels and their toxic effects. This study aims to develop a high-throughput screening method for the therapeutic drug monitoring (TDM) and occupational exposure of cyclophosphamide (CP), an alkylating agent used as chemotherapeutic and immunosuppressive drug. In order to achieve this goal, an immunizing hapten that exposes the cyclophosphamide moiety has been designed for the first time. Antibodies raised against this hapten have been used to develop an indirect competitive ELISA for the quantification of CP with high specificity and low cross-reactivity with some metabolites and other anticancer drugs. The assay obtained reported a LOD of 22 ± 6 nM in serum samples, concentration much below blood CP levels of patients treated with the drug. A new tool for detection and quantification of CP is provided that could be relevant for future pharmacokinetic studies and for therapeutic index improvement., This work has been funded by the ministry of Economy and Competiniveness (MAT2012-38573-C02-01). The Nb4D group is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient: 2017 SGR 1441). CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation 2013-2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Marta Broto wishes to thank the FPI-fellowship (BES-2013-062819) from the Spanish Ministry of Economy and Competitiveness.

An Enzyme Linked Immunosorbent Assay (ELISA) with the ability to detect some of the most widely used penicillins such as penicillin G, amoxicillin and ampicillin has been developed and validated in milk samples to ensure food safety. The assay can achieve limits of detection (0.1 μg L−1) much lower than the MRLs (Maximum Residue Levels) established by the EU (4 μg L−1). The procedure allows the analysis of the samples with a simple and fast treatment of the matrix with biohybrid magnetic particles to hydrolyze the β-lactam ring. A new immunizing hapten has been described and the polyclonal antibodies produced showed high specificity for open ring penicillins and no cross-reactivity with other antibiotic families. The assay has been designed to detect simultaneously both penicillin species in milk, the antibiotics and their hydrolyzed forms. Concerning the open-ringed forms their detection in food samples will be especially important in the next few years because of the illegal use of penicillinase enzymes by farmers to mask the real concentrations of β-lactams in milk.

Cancer is a group of diseases in which abnormal cells grow and divide without control, with the potential to invade other parts of the body. Chemotherapy is a type of treatment that uses chemical agents to treat cancer. These drugs are toxic and produce undesirable adverse drug reactions due to their narrow therapeutic window and highly variable pharmacokinetics, thus, they need to be monitored to establish personalized treatment to achieve maximal efficiency and reduce drug toxicity. Nowadays, therapeutic drug monitoring (TDM) is not routinely used for chemotherapy agents, however, TDM has the potential to improve the clinical benefit of chemotherapy drugs. Tegafur, a prodrug of 5-fluorouracil (5FU), is one of the main anti-cancer drugs used worldwide. Herein, a reproducible and sensitive indirect competitive ELISA has been developed and validated in plasma samples. The assay reports an IC50 of 35.6 nM, reaching a limit of detection of 2.7 nM. It is highly reproducible and does not show cross-reactivity with any related compound. In summary, this assay provides a sensitive, accurate and high throughput analytical method for tegafur quantification in plasma, which fits TDM requirements. © 2017 The Royal Society of Chemistry., This work has been funded by the ministry of Economy and Competiniveness (MAT2012-38573-C02-01). The Nb4D group (formerly Applied Molecular Receptors group, AMRg) is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient : 2014 SGR 1484). CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation 2013-2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Marta Broto wishes to thank the FPI-fellowship (BES-2013-062819) from the Spanish Ministry of Economy and Competitiveness. The Custom Antibody Service (CAbS) is acknowledged for the assistance and support on immunoreagent production and characterization of tegafur antibodies.

A novel approach for therapeutic drug monitoring of oral anticoagulants (OA) in clinical samples is reported, based on a NP-based biobarcode assay. The proposed strategy uses specific antibodies for acenocumarol (ACL) covalently bound to magnetic particles (pAb236-MP) and a bioconjugate competitor (hACL-BSA) linked to encoded polystyrene probes (hACL-BSA-ePSP) on a classical competitive immunochemical format. By using this scheme ACL can be detected in low nM range (LOD, 0.96 ± 0.26, N = 3, in buffer) even in complex samples such as serum or plasma (LOD 4 ± 1). The assay shows a high reproducibility (%CV 1.1 day-to-day) and is robust, as it is demonstrated by the fact that ACL can be quantified in complex biological samples with a very good accuracy (slope = 0.97 and R2 = 0.91, of the linear regression obtained when analyzing spiked vs measured values). Moreover, we have demonstrated that the biobarcode approach has the potential to overcome one of the main challenges of the multiplexed diagnostic, which is the possibility to measure in a single run biomarker targets present at different concentration ranges. Thus, it has been proven that the signal and the detectability can be modulated by just modifying the oligonucleotide load of the encoded probes. This fact opens the door for combining in the same assay encoded probes with the necessary oligonucleotide load to achieve the detectability required for each biomarker target.

The study of adverse drug reactions (ADRs) constitutes a challenge in the area of Medicine. Drugs generate a large number of the total registered hypersensitivity reactions, where penicillins are responsible for more than half of them. In vitro tests in the market are not efficient enough since they lack in sensitivity and specificity. This is the reason why in vivo tests are carried out, with the subsequent danger to the patient’s life. It is essential to discover new β-lactam antigenic determinants to develop more effective detection systems and thus, obtain better explanations of the allergic mechanisms related to these drugs. We propose a strategy based on the use of “peptide probes”, small labeled and chemical active peptides which have been structurally modified for reacting with the β-lactam moiety at different conditions. The probes also contain a biotin group for application in an immunoassay format. Three different amoxicillin adducts have been obtained, purified and characterized by HPLC–MS and NMR techniques. These results have helped us to elucidate and propose a new antigenic determinant for β-lactams, named the “penamidyl” epitope. All the adducts have been validated and evaluated with sera from different penicillin allergic patients by means of a Magneto-ELISA, immunochemical technique that has allowed us to detect specific IgEs in a very high percentage of the serum samples. An immunoassay has been developed, validated and applied as a diagnostic tool for the detection of specific IgEs in the sera of penicillin allergic patients using a new antigenic determinant.

A NP-based biobarcode for C-reactive protein (CRP) quantification in plasma samples is reported for the first time. The assay uses capture antibody functionalized magnetic beads (pAbCRP2-MP), multifunctional oligonucleotide encoded probes modified with a detection antibody (pAbCRP1-ePSP), and a fluorescent DNA microarray. Thus, magnetic beads are added to the sample to form immunocomplexes that will be isolated, to then add the codified particles to form a sandwich complex with both particles and the target protein, subsequently the complexes are treated to release the oligonucleotide codes, which are finally hybridized in a fluorescent DNA microarray. The assay has been implemented to the analysis of plasma samples being able to quantify this biomarker within 900 ng mL −1 to 12500 ng mL −1 with an excellent accuracy (mean of recovery of 99.5 ± 4.2%, N = 3 ). The CRP biobarcode has been used on a small pilot clinical study in which plasma samples from patients suffering different pathologies, most of them related to cardiovascular diseases (CVDs). The samples have been analyzed and the results compared to a reference method demonstrating that the assay can be useful for monitoring this biomarker on patients being suspicious to be under risk of suffering CVDs or other diseases involving inflammatory processes.

Chemotherapy is the use of chemical substances to treat cancer. However, traditional chemotherapeutic agents do not show sufficient specificity for the cancer cells, which means that also healthy cells are strongly affected by these treatments. In parallel, manufacturers and health-care workers are continuously exposed to these cytostatic drugs through the usual procedures. Thus, there is a need to control the real internal dose received by this sector. Bioanalytical techniques may show great potential as tools for establishing a more personalized treatment procedure since therapeutic drug monitoring (TDM) could support clinicians on making and establishing decisions adapted to each patient. Similarly, these bioanalytical procedures may be implemented on those occupational sectors in which risk for exposure to these hazardous drugs exists. In this review, we present the overall situation in that respect and provide information of the bioanalytical techniques reported up to date for the most important cytostatic families. © 2017 Elsevier B.V., The Nb4D group (formerly Applied Molecular Receptors group, AMRg) is a consolidated research group (Grup de Recerca) of the Generalitat de Catalunya and has support from the Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient: 2014 SGR 1484). CIBER-BBN is an initiative funded by the Spanish National Plan for Scientific and Technical Research and Innovation 2013–2016, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was performed as part of a research contract with CETEMMSA Technological Center (part of the EURECAT Technology Center of Catalonia). Marta Broto wishes to thank the FPI-fellowship (BES-2013-062819) from the Spanish Ministry of Economy and Competitiveness.