*3.4. Microfluidics-Based Assays*

In addition to LFA, microfluidic technology has attracted much attention in recent years to detect a variety of analytes. According to the definition provided by Whitesides from Harvard University, "microfluidic is the science and technology of systems that process or manipulate small amounts (10−<sup>9</sup> to 10−<sup>18</sup> L) of fluids, using channels with dimensions of tens to hundreds of micrometers". This technique shows a great potential to control the concentrations of molecules in space and time [82]. High surface-to-volume ratios, small consumption of reagents, prevalence of viscous and capillary forces and laminar flows are the major features of microfluidic-based systems [83]. Based on such properties, microfluidic can be integrated with biosensor technology in order to develop analytical devices with high sensitivity, reproducibility, portability, low-cost, short detection time, and high throughput [84]. Early microfluidic systems were fabricated of silicon and glass. Because of high cost of silicon and fragility of glass, polymer-based devices were then offered in the late 1990s which were cheaper than glass and silicon and provided an extensive range of chemical materials expanded from polydimethylsiloxane (PDMS) to thermoplastics [85,86].

Recently, microfluidic-based assays have attracted a large amount of interest in the detection of mycotoxins. The incorporation of microfluidic system and immunoassay is considered as one of the most popular platforms for detecting mycotoxins with high sensitivity and short detection time. For example, Machado et al. developed a PDMS-based microfluidic immunoassay with four chambers for simultaneous detection of OTA, AFB1, and DON [83]. The competitive immunoassay was developed by the immobilization of BSA-mycotoxin conjugates onto separate chambers (Figure 6a). The first inlet was considered for sample loading. In the presence of the given mycotoxin, the free toxin competed with the toxin-BSA immobilized on the PDMS surface for the specific binding to IgG-HRP conjugate. Therefore, a high concentration of a target free toxin resulted in a low density of IgG-HRP captured by the immobilized BSA-toxin. After addition of TMB, a colorimetric signal was observed which was inversely proportional to the mycotoxin concentration. Smartphone was used to obtain semi-quantitative results. The proposed assay exhibited LODs at <40, 0.1–0.2 and <10 ng·mL−<sup>1</sup> for OTA, AFB1, and ZEN, respectively. Furthermore, the immunoassay was applied for the simultaneous detection of these three mycotoxins in corn samples after a simple sample preparation method. The multiplexed analysis with a relatively low cost and simple operation can be performed in less than 10 min. However, these methods can be simplified by reducing the number of user-intervention steps such as pipetting.

In another microfluidic device, AuNPs were used as colored labels for indicating various concentrations of alternariol monomethyl ether (AME), one of the most frequently occurred Alternaria mycotoxins [87]. Microfluidic chip was fabricated using Norland Optical Adhesive 81 and glass substrate (Figure 6b). AuNPs a conjugated with AME specific mAb and magnetic nanoparticles (MNPs)-BSA-AME conjugates were used as capture probe and competitive antigen, respectively. In the presence of AME, it firstly bound to the AuNPs-mAbs in conjugate pad and micro-mixing channel. Therefore, large numbers of free AuNPs-mAbs-AME conjugates were kept in supernatant after magnetic separation. Then, the supernatant was transferred into immunogold amplification solution containing ascorbic acids as reducing agent and hexadecyltrimethylammonium bromide as a surfactant to stabilize the amplified AuNPs-mAbs. In this solution, the free AuNPs-mAbs-AME conjugates were used as gold seeds for the signal amplification. UV spectroscopy and smartphone imaging APP were used for monitoring of the AuNPs color change. The assay was able to analyze six samples in parallel within 15 min. The fabricated microfluidic immunoassay exhibits LODs at 12.5 pg·mL−<sup>1</sup> and 200 pg·mL−<sup>1</sup> , by UV spectroscopy and smartphone imaging, respectively. It was successfully applied for AME detection in spiked fruit samples. The device can be used for sensitive, rapid, low-cost, and on-site detection of mycotoxins.

**Figure 6.** (**a**) Schematic representation of the microfluidic immunoassay with smartphone data acquisition for multiplexed mycotoxin detection; (**b**) microfluidic immunoassay for AME detection: (**1**) The 3D structural diagram of the fabricated microfluidic chip, (**2**) The schematic of the AME detection using the amplified microfluidic immunoassay, and (**3**) The colorimetric detection by UV spectroscopy and smartphone imaging APP; (**c**) Development of a µPAD using aptamer and for AFB1 detection based on salt-induced aggregation of AuNPs in the presence of analyte. Reproduced from [83,87,88], respectively, with permission.

Paper is an ideal substrate to construct microfluidic devices. It is a good alternative to glass and polymer. Paper-based microfluidic systems were introduced by Whitesides group in 2007 as lab-on-chip (LOC) devices. The paper-based microfluidic devices are cheaper, easier to fabricate, easier to use, easier to dispose, compatible to chemicals/biochemicals used in bio-medical applications and environmentally friendly. Paper segmentation to hydrophobic and hydrophilic regions by hydrophobic materials can provide hydrophilic channels for fluid flow via capillary action and without the need for a pump. However, despite all these advantages, paper-based microfluidic devices are only suitable for semiquantitative rather than quantitative analysis [85,86,89].

− − Although there are a number of well-developed systems for immunoassay in microfluidic (lab-on-chip) format, the use of aptamers in similar devices is on very beginning. Kasoju et al. developed a microfluidic paper-based analytical device (µPAD) for AFB1 detection using aptamer as recognition element [88]. The hydrophobic barriers were developed on the Whatman filter paper using photolithography. Two control zones (negative and positive) and one analyte zone were designed on the paper. Detection was performed based on salt-induced aggregation of AuNPs in the presence of analyte. The aptamer-AuNPs conjugate was adsorbed onto the paper through physical adsorption and AFB1 was allowed to flow over the µPAD. In the presence of AFB1, the aptamer combined with AFB1 and bare AuNPs was aggregated in the presence of NaCl (Figure 6c). The developed assay showed a LOD of 10 nM in spiked samples. The developed µPAD was suitable for rapid (detection time < 1 min), simple, label-free, and on-site detection of mycotoxins.

Representative examples of recent developed microfluidic-based assays for the detection of mycotoxins are reported in Table 5.

*Toxins* **2021**, *13*, 13


**Table 5.** Representative examples of recent developed microfluidic-based assays for the detection of mycotoxins.
