Nuno M. M. Pires

Recent Developments in Optical Detection Technologies in Lab-on-a-Chip Devices for Biosensing Applications

The field of microfluidics has yet to develop practical devices that provide real clinical value. One of the main reasons for this is the difficulty in realizing low-cost, sensitive, reproducible, and portable analyte detection microfluidic systems. Previous research has addressed two main approaches for the detection technologies in lab-on-a-chip devices: (a) study of the compatibility of conventional instrumentation with microfluidic structures, and (b) integration of innovative sensors contained within the microfluidic system. Despite the recent advances in electrochemical and mechanical based sensors, their drawbacks pose important challenges to their application in disposable microfluidic devices. Instead, optical detection remains an attractive solution for lab-on-a-chip devices, because of the ubiquity of the optical methods in the laboratory. Besides, robust and cost-effective devices for use in the field can be realized by integrating proper optical detection technologies on chips. This review examines the recent developments in detection technologies applied to microfluidic biosensors, especially addressing several optical methods, including fluorescence, chemiluminescence, absorbance and surface plasmon resonance.

Integrated optical microfluidic biosensor using a polycarbazole photodetector for point-of-care detection of hormonal compounds

Abstract. A picogram-sensitive optical microfluidic biosensor using an integrated polycarbazole photodiode is developed. The photodetector is mainly composed of the blend heterojunction of poly [N-9′-heptadecanyl-2,7-carbazole-alt-5,5-(4′,7′-di-2-thienyl-2′,1′,3′-benzothiadiazole)] (PCDTBT) and [6,6]-phenyl C71-butyric acid methyl ester (PC70BM) and the poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) as the hole transport layer. Analyte detection is accomplished via a chemiluminescent immunoassay performed in a poly(dimethylsiloxane)-gold-glass hybrid microchip, on which antibodies were immobilized and chemiluminescent horseradish peroxidase-luminol-peroxide reactions were generated. Enhanced sensor response to the chemiluminescent light is achieved by optimizing the thickness of PCDTBT:  PC70BM and PEDOT:PSS. Using the optimized polycarbazole photodiode for detecting the human thyroid-stimulating hormone as the model target, the integrated biosensor demonstrates an excellent linearity in the range of 0.03 to 10  ng/ml with an analytical sensitivity of 68  pg/ml. The sensor response shows high specificity and reproducibility. Hormone detection in clinical samples is further demonstrated and compared with a commercial enzyme-linked immunosorbent assay. The integrated device reported here has potential to detect other hormonal compounds or protein targets. Moreover, the presented concept enables the development of miniaturized, low-cost but highly sensitive optical microfluidic biosensors based on integrated polymer photodetectors with high potential for point-of-care diagnostics.

A mediator embedded micro-immunosensing unit for electrochemical detection on viruses within physiological saline media

To provide a time- and cost-saving alternative to the conventional methods for virus detection in biological media, this work presents an electrochemical micro-immunosensor based on the nickel hexacyanoferrate (NiHCF) redox mediator film coating the interdigitated microelectrodes (IDMEs). By chelation binding with no additional cross-linker, the 6xHis-tagged antibodies were immobilized on a NiHCF film. Secondly, an immunoassay response was enhanced by employing microbeads coated with 6xHis antibody. The electrochemical properties and the stability of the NiHCF film modified IDMEs were evaluated by cyclic voltammetry. The bead-induced impedance variations at the electrode film/electrolyte interface were characterized by electrochemical impedance spectroscopy and verified using FEM simulation. Experiments of virus detection were conducted through targeting the antigens of the vital infectious salmon viruses, such as infectious salmon anaemia virus, infectious pancreatic necrosis virus and salmonid alphavirus subtype 3. The micro-immunosensor exhibited detection limits as low as 10 pg ml−1 and detection sensitivities as high as 57.5 kΩ µM−1 within a physiological saline solution. Tests for multiple antigen–antibody interactions showed good detection specificity, as confirmed by ELISA. By incorporating the microfluidic network, electrochemical impedance micro-immunosensing units can be realized in a fully integrated platform for multiplex virus detection in tissue samples.

Recovery of Cryptosporidium and Giardia organisms from surface water by counter-flow refining microfiltration

As waterborne parasitic cryptosporidiosis and giardiasis outbreaks continue globally, monitoring of Cryptosporidium parvum and Giardia lamblia in surface water continues to be challenging. Lack of non-clogging and high-efficiency methods for recovery of C. parvum oocysts and G. lamblia cysts in environmental water strongly limits the sensitivity of detection methods for these protozoan organisms. In this work, the Counter-Flow Micro-Refinery (CFMR) system was developed by employing the novel counter-flow microfiltration principle to enrich (oo)cysts for subsequent analytical purposes. The CFMR system was constructed with multiple counter-flow concentration units that were arranged into two refining levels. By use of different numbers of units, the CFMR offered an adjustable concentration ratio allowing the concentration of 10 L and 100 L to hundreds of mL with no recirculation processing. With spiked samples, recovery of 81.3% oocysts and 86.2% cysts at a variance of<7% was achieved for concentrations as low as 0.5–100 organisms L−1. The recovery efficiency showed consistent for a wide range of water turbidities as well as different sample volumes. No significant clogging has been observed in the experiments. Moreover, the refining filter was able to enrich and separate oocysts and cysts in water, simultaneously. This work verifies a feasible solution for recovering C. parvum oocysts and G. lamblia cysts in large-volume surface waters. The refining system has potential to be a high-efficiency monitoring tool when combined with proper analytical detection methods.

A cascade-like silicon filter for improved recovery of oocysts from environmental waters

Standard filtration methods have been characterized by poor recoveries when processing large-volume samples of environmental water. A method to pre-remove particulates present in turbid waters would be necessary to enhance recovery of protozoan oocysts. Particulate separation can be achieved by the proposed multiplex particle refining (MPR) system. This system employs multiple counter-flow microfiltration units that are arranged into a cascade-like structure. By use of this design, the target oocysts are pre-concentrated from environmental waters. The performance of the MPR system was investigated using 10-L deionized water and surface water spiked with 100 Cryptosporidium parvum oocysts. A recovery rate of around 85% was obtained for spiked river water. The water samples were processed using high flow rate and a simple filtration protocol. Further experiments were conducted using the MPR as a pre-filter for five commercially available filters. The recovery rates were two- to threefold higher employing the pre-filter than using the filters alone. The merit of the refining system to use different numbers of counter-flow units led to superior oocyst recovery rate for the Filta-Max and Envirochek HV filters, which are approved by the US Environmental Protection Agency. This work demonstrates a feasible tool for improved filtration performance in environmental waters.

Interfacial Impedance Sensor Employing Bio-activated Microbeads and NiHCF-Coated Interdigitated Microelectrodes: A Model Analysis

An impedance sensor based on NiHCF-coated interdigitated microelectrodes is presented as a promising solution for ultra-low level pathogen detection. The micro sensor employs micro-sized beads, which are coated with anti-pathogen specific antibodies, to achieve the high-sensitive detection through the amplified variations of the interfacial impedance. The effect of beads on the microelectrodes solution interface and subsequent impedance change were analyzed and demonstrated by fitting the experimental data to an equivalent electrical circuit model.

Detection of stress hormones by a microfluidic-integrated polycarbazole/fullerene photodetector

A novel photodetector integrated microfluidic system for chemiluminescence (CL) detection is reported. The system incorporates a polycarbazole/fullerene photodiode whose optical characteristics (i.e. dark current, external quantum efficiency and photosensitivity) are described here. Using a CL immunoassay for detecting the stress hormone cortisol, the integrated photodetector achieved a detection sensitivity of 1.775 pA × nM-1 and a detection limit of less than 0.28 nM. The device would be a powerful low-cost alternative to silicon photodiode and photomultiplier tube for bioanalytical assays, with potentially wide-ranging applications within point-of-care diagnostics.

Multiplexed detection of waterborne pathogens with an array of microfluidic integrated high-sensitivity organic photodiodes

It is reported a high-throughput microfluidic system integrating organic photodetectors based on poly(2,7-carbazole) semiconducting polymer. The system reported here showed detection limits of 105-106 cells/mL for the chemiluminescence detection of two bacterial cells. The detection of viral particles as low as 10-4 μg/mL was also shown. High specificity and ability to perform multiplexed analysis in an integrated lab-on-a-chip device was demonstrated.

An infrared radiation based thermal biosensor for enzymatic biochemical reactions

In this paper, a thermal biosensor based on the infrared radiation energy is proposed for calorimetric measurement of biochemical reactions. Having a good structure design combined with MEMS technology as well as employing the Si/SiGe quantum well sensing material with a high TCR and low 1/f noise, the sensor shows potentials to be high sensitive and real-time. The urea enzymatic reaction was tested to verify the performance of sensor, which demonstrates a linear detection range from 0.5mM to 150mM and a relative standard deviation less than 1%. For the sensor fabrication, wafer-level transfer bonding is a key process, which makes the integration of quantum well material and a free standing structure possible. It reduces the heat loss from the sensor to the surrounding environment.

Detection of pathogens using bio-activated nickel hexacyanoferrate film coated interdigitated microelectrodes

In the present work, the microfabricated impedance sensing array (MISA) unit, based on interdigitated microelectrodes (IDMEs) coated with nickel hexacyanoferrate (NiHCF) film, is presented as an alternative solution to detect pathogens. The NiHCF film, which provide a [Fe(CN)6]3-/4- probe-free redox reaction, was functionalized with hexahistine tags (6×His) antibodies with which they have the high-affinity. Variation in the interfacial impedance caused by bio-activated beads was measured to detect targeted Infectious Salmon Anaemia Virus (ISAV) antigen. A detection limit of 10pg/ml was demonstrated, which indicates that the MISA unit is promising to detect pathogens at early-stage of the infection.