Ruben R.G. Soares

Partitioning in aqueous two-phase systems : Analysis of strengths, weaknesses, opportunities and threats

For half a century aqueous two‐phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single‐use, flexible biomanufacturing has led to industrial re‐evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi‐stage operations and in single‐step purification processes.

DNA aptamer-based sandwich microfluidic assays for dual quantification and multi-glycan profiling of cancer biomarkers

Two novel sandwich-based immunoassays for prostate cancer (PCa) diagnosis are reported, in which the primary antibody for capture is replaced by a DNA aptamer. The assays, which can be performed in parallel, were developed in a microfluidic device and tested for the detection of free Prostate Specific Antigen (fPSA). A secondary antibody (Aptamer-Antibody Assay) or a lectin (Aptamer-Lectin Assay) is used to quantify, by chemiluminescence, both the amount of fPSA and its glycosylation levels. The use of aptamers enables a more reliable, selective and controlled sensing of the analyte. The dual approach provides sensitive detection of fPSA along with selective fPSA glycoprofiling, which is of significant importance in the diagnosis and prognosis of PCa, as tumor progression is associated with changes in fPSA glycosylation. With these approaches, we can potentially detect 0.5 ng/mL of fPSA and 3 ng/mL of glycosylated fPSA using Sambucus nigra (SNA) lectin, both within the relevant clinical range. The approach can be applied to a wide range of biomarkers, thus providing a good alternative to standard antibody-based immunoassays with significant impact in medical diagnosis and prognosis.

Aqueous two-phase systems for enhancing immunoassay sensitivity: Simultaneous concentration of mycotoxins and neutralization of matrix interference

Immunoassays have a broad application range, from environmental and food toxicology to biomedical analysis, providing rapid and simple methods for analyte quantification. Immunoassays, however, are often challenging at nM and sub nM concentrations and are affected by detrimental matrix interference effects, as is the case of the detection of ochratoxin A (OTA) and Aflatoxin B1 (AFB1). These are widespread mycotoxins found in food and feed, with serious potential implications for human health. This work demonstrates the use of polymer-salt aqueous two phase systems (ATPSs) for the simultaneous concentration of mycotoxins and neutralization of matrix interference. In particular, polyethylene glycol (PEG)-phosphate salt ATPSs were used to enhance the detection sensitivity of OTA and AFB1 in wines and beer by an indirect competitive ELISA. Using this methodology it was possible to quantify both analytes spiked in red wine with limits-of-detection (LoD) down to 0.19 ng/mL and 0.035 ng/mL, respectively, with results comparable to those obtained using solutions of toxins in phosphate buffered saline (PBS) buffer (0.7 ng/mL and 0.009 ng/mL, respectively). Furthermore, a very low matrix-to matrix variability was observed, with LoD and half inhibitory concentration (IC50) values of 5.17 ± 1.08 and 33.2 ± 3.5 ng/mL (±SD) obtained in the detection of OTA spiked in red and white wines, beer or PBS buffer. These results indicate the potential of ATPS as a fast and simple concentration step and in providing matrix-independent analyte quantification for enhanced immunoassay sensitivity below regulatory levels.

A point-of-use microfluidic device with integrated photodetector array for immunoassay multiplexing: Detection of a panel of mycotoxins in multiple samples.

For a point-of-use analytical device to be successful in real-world applications, it needs to be rapid, simple to operate and, ideally, able to multiplex the detection of several analytes and samples. Mycotoxin detection in food and feedstock in particular has become increasingly relevant as these toxins, such as ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON), are subject to strict regulations and recommendations in the European Union. A novel, simple, negative pressure-driven device with manually operated magnetic valves was developed and the simultaneous immunodetection of these three mycotoxins was demonstrated via the laminar flow patterning of probes in an area of ≈0.12mm2 and subsequent chemiluminescence generation via HRP-labeled antibodies. The three mycotoxins were detected in less than 20min at concentrations of 100ng/mL for OTA and DON and 3ng/mL for AFB1, spiked in a sample under analysis and simultaneously compared to a toxin-free reference and a standard contaminated with critical target concentrations. The on-chip optical detection was performed in a single acquisition step by integrating a microfabricated array of 25×25µm2 hydrogenated amorphous silicon (a-Si:H) photosensors below the microfluidic chip. The device presented in this work is simple and effective for point-of-use multiplexing of immunoassays and was applied in this work to the screening of mycotoxins.

Multiplexed capillary microfluidic immunoassay with smartphone data acquisition for parallel mycotoxin detection

The field of microfluidics holds great promise for the development of simple and portable lab-on-a-chip systems. The use of capillarity as a means of fluidic manipulation in lab-on-a-chip systems can potentially reduce the complexity of the instrumentation and allow the development of user-friendly devices for point-of-need analyses. In this work, a PDMS microchannel-based, colorimetric, autonomous capillary chip provides a multiplexed and semi-quantitative immunodetection assay. Results are acquired using a standard smartphone camera and analyzed with a simple gray scale quantification procedure. The performance of this device was tested for the simultaneous detection of the mycotoxins ochratoxin A (OTA), aflatoxin B1 (AFB1) and deoxynivalenol (DON) which are strictly regulated food contaminants with severe detrimental effects on human and animal health. The multiplexed assay was performed approximately within 10min and the achieved sensitivities of<40, 0.1-0.2 and<10ng/mL for OTA, AFB1 and DON, respectively, fall within the majority of currently enforced regulatory and/or recommended limits. Furthermore, to assess the potential of the device to analyze real samples, the immunoassay was successfully validated for these 3 mycotoxins in a corn-based feed sample after a simple sample preparation procedure.

Miniaturization of aqueous two-phase extraction for biological applications: From micro-tubes to microchannels.

Aqueous two‐phase extraction (ATPE) is a biocompatible liquid‐liquid (L‐L) separation technique that has been under research for several decades towards the purification of biomolecules, ranging from small metabolites to large animal cells. More recently, with the emergence of rapid‐prototyping techniques for fabrication of microfluidic structures with intricate designs, ATPE gained an expanded range of applications utilizing physical phenomena occurring exclusively at the microscale. Today, research is being carried simultaneously in two different volume ranges, mL‐scale (microtubes) and nL‐scale (microchannels). The objective of this review is to give insight into the state of the art at both microtube and microchannel‐scale and to analyze whether miniaturization is currently a competing or divergent technology in a field of applications including bioseparation, bioanalytics, enhanced fermentation processes, catalysis, high‐throughput screening and physical/chemical compartmentalization. From our perspective, both approaches are worthy of investigation and, depending on the application, it is likely that either (i) one of the approaches will eventually become obsolete in particular research areas such as purification at the preparative scale or high‐throughput screening applications; or (ii) both approaches will function as complementing techniques within the bioanalytics field.

A simple method for point-of-need extraction, concentration and rapid multi-mycotoxin immunodetection in feeds using aqueous two-phase systems

The rapid detection of mycotoxins in feed samples is becoming an increasingly relevant challenge for the food production sector, in order to effectively enforce current regulations and assure food and feed safety. To achieve rapid mycotoxin detection, several biosensing strategies have been published, many reaching assay times of the order of a few minutes. However, the vast majority of these rely on sample preparation based on volatile organic solvents, often comprising complex multi-step procedures and devoid of clean-up and/or concentration effects. Here, a novel sample preparation methodology based on a green, non-toxic and inexpensive polyethylene glycol-sodium citrate aqueous two-phase system is reported, providing single-step extraction and concentration of three target mycotoxins within 20min: aflatoxin B1 (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON). With point-of-need applications in mind, the extraction procedure was optimized and validated using a rapid multi-toxin microfluidic competitive immunoassay. The assay was successfully tested with spiked complex solid matrices including corn, soy, chickpea and sunflower-based feeds and limits of detection of 4.6ngg-1±15.8%, 24.1ngg-1±8.1% and 129.7ngg-1±53.1% (±CV) were obtained in corn for AFB1, OTA and DON, respectively. These sensitivities are fit-for-purpose at the required regulatory and recommended limits for animal feed, providing an effective and safe semi-quantitative mycotoxin analysis that can be performed in the field.

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.

The application of microbeads to microfluidic systems for enhanced detection and purification of biomolecules

This paper describes microbead-based microfluidic systems. Several aspects of bead assays in microfluidics make them advantageous for bioassays in simple microchannels, including enhanced surface-to-volume ratio, improved molecular recognition reaction efficiency, and the wide range of surface functionalization available with commercial microbeads. Two-level SU-8 molds are used to fabricate PDMS microchannels that can hydrodynamically trap different types of microbeads, with characteristic dimensions of tens of microns. The use of these microbead-based microfluidic systems in the biosensing of antibodies, toxins and nucleic acids, as well as in antibody purification will be presented and discussed in this paper.

Lab-on-chip systems for integrated bioanalyses

Biomolecular detection systems based on microfluidics are often called lab-on-chip systems. To fully benefit from the miniaturization resulting from microfluidics, one aims to develop ‘from sample-to-answer’ analytical systems, in which the input is a raw or minimally processed biological, food/feed or environmental sample and the output is a quantitative or qualitative assessment of one or more analytes of interest. In general, such systems will require the integration of several steps or operations to perform their function. This review will discuss these stages of operation, including fluidic handling, which assures that the desired fluid arrives at a specific location at the right time and under the appropriate flow conditions; molecular recognition, which allows the capture of specific analytes at precise locations on the chip; transduction of the molecular recognition event into a measurable signal; sample preparation upstream from analyte capture; and signal amplification procedures to increase sensitivity. Seamless integration of the different stages is required to achieve a point-of-care/point-of-use lab-on-chip device that allows analyte detection at the relevant sensitivity ranges, with a competitive analysis time and cost.