Microfluidic devices for glycobiomarker detection in cancer.

During oncogenesis, several alterations occur within cells, one of them being the abnormal glycosylation of proteins, resulting in the formation of glycoproteins with aberrant glycan structures, which can be secreted into the blood stream. Their specific association to tumour cells makes them useful indicators (biomarkers) of the oncogenic process and their detection in blood can be employed in different stages of tumour development for early detection, prognosis and therapeutic drug monitoring. Due to the importance of detecting cancer-associated glycoproteins with aberrant glycosylation in blood or serum, analytical methodologies with improved performance are required to ameliorate the laboratorial tests currently used for the detection of these analytes. Microfluidics was created to facilitate the implementation of simple and point-of-care analysis, away from a centralized laboratory. The massive use of microfluidic systems in clinical settings can be seen in pregnancy tests and diabetes control, for example. But what about other clinical domains, such as the detection of glycoproteins with aberrant glycans secreted by tumour cells? Are microfluidic systems helpful in this case? This review analyses the requirements of a microfluidic assay for the detection of low-abundant blood/serum cancer-associated glycoproteins with abnormal glycans and the progresses that have been made in the last years to develop integrated microfluidic devices for this particular application. The diverse microfluidic systems found in literature present, in general, the same analytical performance as the conventional assays but have additional advantages, namely a reduction in assay times, a decrease of sample and reagent consumption and lower costs. The review will also focus on the improvements that are still needed for better biosensing of this type of cancer biomarkers using microfluidic devices.

Lectin biosensors in cancer glycan biomarker detection.

Cancer has high incidence and it will continue to increase over the next decades. Detection and quantification of cancer-associated biomarkers is frequently carried out for diagnosis, prognosis and treatment monitoring at various disease stages. It is well-known that glycosylation profiles change significantly during oncogenesis. Aberrant glycans produced during tumorigenesis are, therefore, valuable molecules for detection and characterization of cancer, and for therapeutic design and monitoring. Although glycoproteomics has benefited from the development of analytical tools such as high performance liquid chromatography, two-dimensional gel and capillary electrophoresis and mass spectrometry, these approaches are not well suited for rapid point-of-care (POC) testing easily performed by medical staff. Lectins are biomolecules found in nature with specific affinities toward particular glycan structures and bind them thus forming a relatively strong complex. Because of this characteristic, lectins have been used in analytical techniques for the selective capture or separation of certain glycans in complex samples, namely, in lectin affinity chromatography, or to characterize glycosylation profiles in diverse clinical situations, using lectin microarrays. Lectin-based biosensors have been developed for the detection of specific aberrant and cancer-associated glycostructures to aid diagnosis, prognosis and treatment assessment of these patients. The attractive features of biosensors, such as portability and simple use make them highly suitable for POC testing. Recent developments in lectin biosensors, as well as their potential and pitfalls in cancer glycan biomarker detection, are presented in this chapter.

Detection of the cancer-associated T antigen using an Arachis hypogaea agglutinin biosensor.

An impedimetric biosensor was developed for the selective detection of the cancer-associated T antigen, using the lectin from Arachis hypogaea (peanut agglutinin, PNA) as the recognition element. The increase in the biosensor's impedance after sample incubation was indicative of lectin recognition and complex formation between PNA and glycoproteins containing T antigen. When using asialofetuin as model glycoprotein, a minimum amount of 100 ng of glycoprotein could be detected, generating an increase in impedance of 7.2%. Albumin did not cause interference in the detection of T-carrying glycoproteins up to a concentration of 0.01 mg ml-1. The biosensor was used to evaluate the T-antigen expression in serum samples and was able to discriminate between control samples (of individuals without cancer) and case samples from patients with diverse types of carcinomas (skin, colon, breast, prostate, stomach, kidney, lung, liver and rectum) in which an increase in the expression of T antigen is well-known. The same samples were analyzed with a Vicia villosa agglutinin biosensor that has specificity for the cancer-associated Tn antigen, to compare the expression of both antigens in the diverse carcinomas. The results were different for both biosensors, confirming that the use of different lectins allows to monitor different antigen expression. Furthermore, combining different lectins, glycosylation profiles for each carcinoma type can be obtained. This work demonstrates the feasibility of employing PNA to selectively recognize the T epitope in glycoproteins and the proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated T antigen in serum samples.

Lectin-based biosensors as analytical tools for clinical oncology.

The review focus on the use of lectin-based biosensors in the oncology field, and ponders the potentialities of using these devices as analytical tools to monitor the levels of cancer glycobiomarkers in biological fluids, helping in the diagnosis, prognosis and treatment assessment. Several examples of lectin-based biosensors directed for cancer biomarkers are described and discussed, and their potential application in the clinic is considered, taking into account their analytical features, advantages and performance in sample analysis. Technical and practical aspects in the construction process, which are specific for lectin biosensors, are debated, as well as the requirements in sample collection and processing, and biosensor validation. Today's challenges for real implementation of these devices in the clinic are presented, along with the future trends in the field.

Cancer serum biomarkers based on aberrant post-translational modifications of glycoproteins: Clinical value and discovery strategies.

Due to the increase in life expectancy in the last decades, as well as changes in lifestyle, cancer has become one of the most common diseases both in developed and developing countries. Early detection remains the most promising approach to improve long-term survival of cancer patients and this may be achieved by efficient screening of biomarkers in biological fluids. Great efforts have been made to identify specific alterations during oncogenesis. Changes at the cellular glycosylation profiles are among such alterations. The "glycosylation machinery" of cells is affected by malignant transformation due to the altered expression of glycogens, leading to changes in glycan biosynthesis and diversity. Alterations in the post-translational modifications of proteins that occur in cancer result in the expression of antigenically distinct glycoproteins. Therefore, these aberrant and cancer-specific glycoproteins and the autoantibodies that are produced in response to their presence constitute targets for cancer biomarkers' search. Different strategies have been implemented for the discovery of cancer glycobiomarkers and are herein reviewed, along with their potentialities and limitations. Practical issues related with serum analysis are also addressed, as well as the challenges that this area faces in the near future.

Construction and validation of a Sambucus nigra biosensor for cancer-associated STn antigen.

A label-free electrochemical impedance spectroscopy biosensor for selective detection and discrimination of the cancer-associated sialyl-Tn (STn) antigen was developed by using Sambucus nigra agglutinin type I (SNA-I) as the recognition element. The SNA-I biosensor was constructed by immobilizing the lectin on screen-printed gold electrodes. The formation of a complex between SNA-I and STn-containing glycoproteins (transferrin and bovine submaxillary mucin) was monitored by measuring the impedance increase of the biosensor. The increase in electron transfer resistance was linearly proportional to the concentration of the glycoproteins up to 70 ng of transferrin and 40 ng of bovine submaxillary mucin, with a limit of detection of 20 ng for transferrin. Albumin, the most abundant serum protein, did not interfere in the detection of the STn-glycoproteins up to a concentration of 0.2 mg ml(-1). The developed lectin-based biosensor was used to evaluate the STn-expression in serum samples and discriminate samples from healthy individuals and patients with different types of malignant tumors, mostly carcinomas, where the increased expression of STn aberrant glycans is well established. This work demonstrates the feasibility of employing SNA-I to selectively recognize the STn epitope in glycoproteins and the use of the constructed biosensor was effective in the analysis of serum samples with the ability to discriminate in a fast way between cancer and healthy status. The proposed biosensor could be used for high-throughput, label-free profiling of the cancer-associated STn glycan expression in serum for diagnosis and therapy monitoring.

Voltammetric determination of food colorants using a polyallylamine modified tubular electrode in a multicommutated flow system.

This work describes the construction of a polyallylamine modified tubular glassy carbon electrode and its application in the electroreduction of food azo colorants (tartrazine, sunset yellow and allura red) by square wave voltammetry. The electrode modification prevented the surface fouling and, simultaneously, enhanced the analytical signal intensity. The developed unit was coupled to a multicommutated flow system which, given the complexity of samples, was designed to allow the implementation of the standard additions method in an automatic way, using only one standard solution. The described method presented a linear range up to about 2.0x10(-4)moll(-1) for the referred colorants, with a detection limit of 1.8x10(-6)moll(-1) for tartrazine, 3.5x10(-6)moll(-1) for sunset yellow and 1.4x10(-6)moll(-1) for allura red. The method was applied in the analysis of these colorants in several food samples, and no statistically significant difference between the results obtained by the proposed and the comparative method (HPLC) was found, at a 95% confidence level. Repeatability in the analysis of samples (expressed in R.S.D.) was about 3% (n=10).

Modified tubular electrode in a multi-commutated flow system: determination of acetaminophen in blood serum and pharmaceutical formulations.

This work describes the construction of a Nafion coated glassy carbon tubular electrode and its use, coupled to a multi-commutated flow system, for the voltammetric determination of acetaminophen in serum and pharmaceutical formulations. The modification of the electrode enhanced the analytical signal intensity and, simultaneously, prevented the electrode surface fouling. The multi-commutated system conferred high versatility to the manifold, allowing it to be easily adjusted to each determination without the need to introduce any physical reconfiguration. The on line enzymatic hydrolysis of acetaminophen, giving rise to 4-aminophenol, allowed the problem of interferences resulting from the oxidation of the matrix serum constituents to be overcome. The method presented a linear range up to 5.0 x 10(-4) mol l(-1) with a detection limit of 1.7 x 10(-5) mol l(-1), a sampling rate of 24 determinations per hour and a repeatability (expressed in relative standard deviation) always lower than 3%. The method was applied to serum samples and pharmaceutical formulations and no statistically significant difference between the results obtained by the proposed method and by the reference methods was found, for a confidence level of 95%.