key: cord-0060199-atsqmp2s
authors: Espinosa-Hernandez, Michelle Alejandra; Reveles-Huizar, Sofia; Hosseini, Samira
title: Bio-microelectromechanical Systems (BioMEMS) in Bio-sensing Applications-Colorimetric Detection Strategies
date: 2020-08-14
journal: BioMEMS
DOI: 10.1007/978-981-15-6382-9_2
sha: 8a5f08da262826dfcd02bf4f45aafb1feccc3297
doc_id: 60199
cord_uid: atsqmp2s

Colorimetric detection is one of the main strategies used in biosensors due to the inexpensive, rapid, and easy readout. Among the main types of colorimetric biosensors are paper-based, lab-on-chip, lab-on-compact disk, and other alternative devices. In this chapter, some of the recent examples of BioMEMS are provided including DNA-detecting sensors based on acpcPNA-induces NP aggregation, neuropeptide Y-detecting ELISA platforms, streptococcus pneumonia and E. coli-detecting biosensors incorporating nucleus acid extraction, smartphone-based devices for biomarkers detection in sweat and saliva, and glass nanofiber-based biosensors for the detection of cholinesterase inhibitors. This chapter provides a thorough review of the latest advancements in of bio-microelectromechanical systems (BioMEMS) that operate based on the principle of colorimetric detection. A detailed comparison between the fabrication and operation of these devices along with the advantages and disadvantages of each technique are also included in this chapter.

Li (2016) Paper-based analytical device (PAD); Pyrrolidinyl peptide nucleic acid (acpcPNA); Silver nanoparticles ( colored substance, light wavelengths are absorbed in different proportions (Fuwa and Vallee 1963) . The intensity of the resultant color will be proportional to the concentration of the measured analyte and the amount of absorbed light will be proportional to the intensity of the color (Ricci et al. 1994 ). Colorimetric analysis is applicable to detect the presence of organic and inorganic compounds, making it a suitable option for biosensors. Some applications for colorimetric detection devices include hand-held bio-diagnostics, point-of-care diagnostics, and naked-eye detection. Current chapter focuses on the fabrication of microfluidic, paper-based, or polymer-based platforms based on this common detection strategy.

One of the major problems in healthcare nowadays is accessibility. Many people worldwide have limited to no access to laboratories or hospitals, hence, in order to reach these communities, smaller portable devices with the same accuracy are needed. Among these developments, the paper-based analytical devices (PAD) attract a great deal of attention. PAD devices have proven to be the inexpensive, simple, portable, and disposable. Likewise, they are easy to use, make complicated readout equipment unnecessary, and produce semi quantitative results (Teengam et al. 2017 ) in a short amount of time (Murdock et al. 2013) . For that reason, PADs are being used to diagnose diseases via DNA and/or RNA recognition (Teengam et al. 2017) , monitor human activity (Murdock et al. 2013) , detect nucleic acids (Choi 2016) , sense pH in sweat and/or saliva (Oncescu et al. 2013) , and recognize cholinesterase inhibitors , thus, making such devices favorable for a wide range of applications including medical (Teengam et al. 2017) , military (Murdock et al. 2013) , nutrition (Choi 2016) , biochemical (Oncescu et al. 2013) , and nerve chemical warfare . Below, several examples of PADs are presented with a specific focus on biosensing application. In addition to the previously stated benefits of paper-based devices, μPADs enhance point of care for detecting diseases (Sanjay et al. 2016) , biomolecules (Li et al. 2018; Gabriel et al. 2017) , and antibiotics (Nilghaz and Lu 2019). They offer inexpensive, simple, eco-friendly, portable and quick bioanalysis (Nilghaz and Lu 2019) . Additionally, due to their small size, these devices provide a greater surface to volume ratio, improving the immobilization of proteins through processes such as enzymelinked immunosorbent assay (ELISA) integrated into this platform, and other biological agents (Sanjay et al. 2016) . Many variations and additions can be made to μPADs in order to enhance its properties. For example, cotton, being a similar material to paper and providing the same advantages as well as being stronger and more durable, becomes an option for embedding into daily wearable products (Nilghaz et al. 2015) . Also, by using chemical vapor deposition (CVD) instead of wax printing, more complex detection chambers and a higher degree of reliability for colorimetric detection result can be achieved (Lam et al. 2017) . Some of the latest examples of the paper-based BioMEMS used for colorimetric detection are provided here. Teengam et al. (2017) produced a paper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid-induced nanoparticle aggregation in order to have a simple and quantitative means of detecting diseases such as Middle East Respiratory Syndrome (MERS), Tuberculosis (TB), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and Human Papillomavirus (HPV). To create the device, a multiplex colorimetric PAD with a derived backbone from D-proline/2aminocyclopentanecarboxylic acid (acpcPNA), silver nanoparticles (AgNPs) and a paper-based multiplex DNA sensor were used. The actual PAD was made through a wax-printing technique, and the sensor was based on an origami concept made of two layers, as can be seen in Fig. 2 .1. The base consisted of four wax-defined channels extending outward from the sample reservoir (6 mm i.d.) and the top layer which had four detection and control zones (4 mm i.d.). The sample reservoir at the top was fully punched to the bottom layer, and the top was folded over. Together with a polydimethylsiloxane (PDMS) lid and a 6 mm diameter hole over the reservoir, they were held together. Eight 4 mm holes and control zones were aligned to maintain a constant pressure across the surface where the acpcPNA probe and AgNPs solution were included. The sample solution was added to the sample reservoir where it flows through the channels to wet the colorimetric detection zones. These zones were obtained by placing 10 μL of AgNPs in 0.1 M phosphate buffer saline (PBS) Fig. 2 .1 Design and setup of paper-based multiplex DNA sensor (Teengam et al. 2017) pH 7.4 with a ratio of 5:1 (AgNPs:PBS). For the colorimetry, special acpcPNA probes were designed and fabricated to detect synthetic oligonucleotide targets with sequences in MERS-CoV, MTB, and HPV (DNA com ). The intensity of the color was compared to a single-base mismatch (DNA m1 ), two-base mismatch (DNA m2 ), and DNA nc sequences. In the presence of the DNA com the intensity decreased and was unaffected by the mismatched and noncomplementary targets. There was a high selectivity to single-base mismatch, two-base mismatch, and noncomplementary target DNA.

Paper-based enzyme-linked immunosorbent assays (P-ELISAs) were created by Murdock et al. (2013) in order to measure biomolecules concentrations. In comparison to regular ELISAs, P-ELISAs are faster to make and obtain results. Neuropeptide Y was the point of interest as it is related to regulating stress, anxiety, fear, and overall sympathetic nervous system activity (Eaton et al. 2007; Heilig 2004) . The target of this study was diagnosing Post Traumatic Syndrome and identifying the difference between more exposed soldiers from beginners. The platform of the P-ELISA consisted of a wax-printed 96 (12 by 8 arrays of circular test zones)-microzone paper plate, designed on Office PowerPoint according to the standard Costar 96-well microtiter plate (Murdock et al. 2013) . The wax covered the areas between wells, leaving the 5.56 mm diameter wells hollowed. 3 μL of target solution was inserted into each well, followed by blocking and addition of antibody and incubation. Each test zone was 3 mm in diameter and needed 1.5 μL to be damp. Gold nanoparticles (AuNPs) of 16 nm were added to poly(ethylene glycol) (PEG) and cleaned by centrifugation and buffer exchanges. Anti-rabbit IgG antibodies were linked to the carboxylic end of the PEG. For the P-ELISA, the same process was followed except for the AuNP-IgG which was included instead of the conventional antibody through a silver enhancement kit (Ted Pella/BBInternational). The operation was tested by means of a standard 96-well plate-based ELISA procedure detecting rabbit IgG with a colorimetric substrate ( Fig. 2.2) . The reason behind using wax-printed paper-based was to create an inexpensive method for carrying out biomolecular assays in small volumes. The device allowed the limit of detection (LOD) to be reduced from nano to picomolar scale. Additionally, it permitted a broader range of colorimetric substances be used since the dynamic imaging ranges through conversion to grayscale. Hence, a portable device camera can be used instead of laboratory equipment to carry out the read out. The device increased the number of samples analyzed per dollar unit typically spent on diagnosis while equally increased the number of patients helped. This device holds great promises for its application in remote or resource-limited areas. Choi et al. (2016) developed an integrated paper-based sample-to-answer biosensor for nucleic acid extraction and amplification at the POC. This provided a new view to the operation of paper-based devices as the readout could be done through visual detection or quantification using a smartphone. The device was credited as a high performance microdevice since the colorimetric detection by the naked eye could be performed within an hour. Following this strategy, a battery-powered heating device was introduced to amplify the nucleic acid in POC, which, coupled with the assay, offered a rapid target detection. A Fast Technology Analysis (FTA)

IgG concentration using Au NP-silver enhancement procedure. b Enzyme-free P-ELISA assay (Murdock et al. 2013) card and glass fiber were added to a lateral flow strip for nucleic acid extraction and amplification. First, the paper matrices were separated by hydrophobic polyvinyl chloride (PVC) layers, or valves, shown in Fig. 2. 3. These valves controlled the flow from the nucleic acid extraction to the amplification zone and lateral flow strip. The device had three microfluidic layers in total: first layer for the injection holes and microfluidic channels for reagent transportation, second held the micropatterns for DNA extraction and amplification, and third incorporated a lateral flow strip for the colorimetric detection. The micropatterns were obtained from polycarbonate (PC) sheets and the microstructures from a poly(methyl methacrylate) (PMMA) sheet using a CNC milling machine. A double-sided adhesive film was used to assemble these layers. Lastly, the lateral flow strips were placed between the last two layers. The heating device was included to the integrated biosensor for very sensitive and specific loop-mediated isothermal amplification (LAMP). The bacteria were lysed outside of the device before being introduced to the inlet. This was filled in a channel by capillary forces and the debris in the microbead-bed channel flashed out to the waste chamber. Subsequently, the washing buffer erupt, forcing the solution to go through the microbeads and carry the adsorbed DNA and reaction mix into the LAMP chamber. The reaction is performed at 66°C for 50 min and later loaded onto the lateral flow strip through the connecting channels. The developed biosensor in this study detected Escherichia coli (E. coli) in several food types with LOD as low as 10 (Choi 2016) to 1000 CFU mL −1 and Streptococcus pneumonia in blood samples. Hence, proving the potentials in medical, food safety and environmental applications.

Due to the worldwide use of smartphones, Oncescu et al. (2013) created a health accessory for colorimetric detection of biomarkers in sweat and saliva based on the fact that the pH in saliva can be used to point out enamel decalcification and the pH in sweat helps indicate dehydration. The device is a noninvasive real-time analysis with disposable test strips that is connected to the phone. The pH sensing system consisted of a smartphone case, application, and test strips. The case has a slot where the strips could be inserted to be analyzed and was 3D printed from opaque Vera black material to isolate the strip form variable external light. The colorimetric analysis took place with the help of the phone's camera and a storage compartment for up to six strips. The strips were 3D printed to include an indicator strip, reference strip, and a flash diffuser. The first strip was 9 × 4 mm and was cut out from a pHydrion Spectral 5.0-9.0 plastic pH indicator strip for sweat testing and a 1.0-14.0 strip for saliva. The second strip was made of white plastic material and its purpose was to detect changes in white balance on the camera by the different light conditions or user error. The latter strip was a 2 mm thick membrane of PDMS used to minimize variations in the reading for different lighting conditions, allowing light from the camera's flash to diffuse and illuminate the posterior part of the test strip equally. Additional to the hardware, a software app was made for image acquisition and processing, and data storage and manipulation. The system as a whole, shown in Fig. 2.4 , worked by first loading the app and selecting the test strip of different biomarker tests. Once the app Fig. 2.4 a Device with the test strip being removed from storage compartment, b Obtaining sweat and saliva samples, c inserting test strip into optical system for reading, d pH analysis with test strip inserted Oncescu et al. (2013) loads the calibration data and user interface, the test strip was to be inserted into the case and touch "Analyze" on the screen. The app takes a flashed image of the strip and then is categorized by color.

Matějovský and Pitschmann have created an addition from glass nanofibers to the Detehit biosensor for cholinesterase inhibitors . These inhibitors interfere with the nerve impulses cholinergic transfer mechanism. The biosensor was made based on the cholinesterase reaction based on enzymatic degradation of the substrate to obtain the appropriate acid or thiocholine. The Detehit biosensor was a detection ribbon based on hardened PVC and contains a detection fabric with immobilized and stabilized acetylcholinesterase (AChE), and a cellulose paper strip with acetylthiocholine iodide (ATChI) and Ellman's reagent, shown in Fig. 2 .5. For its operation, the detection fabric ought to be moist and exposed to air, by placing the fabric in contaminated water or pressing a wet detection zone against the test surface. The cellulose strip was squeezed with the exposed detection fabric, making the color change visible to be analyzed. If the white detection fabric changed to yellow, there were no inhibitors, however, if the color did not change, there were inhibitors present. This is depicted in Fig. 2.6a . However, because the color differentiation was sometimes difficult, a new substrate carrier made of glass nanofibers and using a chromogenic reagent (Ellman's reagent) was developed to increase the intensity of the yellow color. The two were compared by having Ellman's reagent combined with the ATChI and an alternate butyrylthiocholine iodide (BuTChI) and testing the new device with AChE and butyrylcholinesterase (BuChE). It was a 10 cm long by 1 cm wide plastic strip, having an indication fabric on one end with an immobilized enzyme covering about 1 cm 2 , while the carrier was at the other end impregnated with a substrate and an indicator, also measuring 1 cm 2 , as seen in Fig. 2.6b . The detection fabric was made by impregnating a white cellulose fabric with a solution containing the enzyme (AChE tissue, AChE, or BuChE) with total activity of 21 nkat/mL, 5% of dextran, and 2% of anionic tenside, in a phosphate buffer solution with a 7.6 pH to be later dried at 25°C for 24 h. The glass and cellulose papers were impregnated with a 4.3 mmol/L solution of Ellman's reagent and with 6 mmol/L of ATChI or BuTChI in ethanol. The indicator paper was dried for 6 h at 25°C. A blank test was performed to compare, and it showed that the glass nanofibers provided an augmented color effect, as is shown in Fig. 2.7.  Fig. 2 .7 Development of color change in the detection zone in (a) filter glass paper, b filter cellulose paper Recent examples of micro paper-based devices have been composed of 96 microfluidic wells (Sun et al. 2010; Sapsford 2009; Kai 2012) . Sanjay et al. (2016) created a 56-microwell paper/PMMA hybrid microfluidic microplate for detection of infectious diseases and other bioanalytes, such as Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg). The chip was laser cut based on the Adobe Illustrator design. In the mask-less laser ablation, the PMMA substrate was placed on a stage. The choice of using porous paper for the flow-through microwells in the PAD allowed the antibodies and antigens to be quickly immobilized, washed effectively, and avoid complicated surface modifications. The microfluidic microplate was composed of three PMMA layers, as seen in Fig. 2 .8a. The first layer (Fig. 2.8bI) consists of an inlet reservoir (Fig. 2.82 .8b1) and fluid distribution channel (Fig. 2.8b3 ) which was used for fluid delivery. It delivered the assay reagents to multiple microwells, which avoids manual pipetting and costly machinery, and forms the cover for the microwells in the following layer. Every channel in the top layer was connected to different inlet reservoirs and delivered the reagents to 7 microwells to the next layer. The second layer (Fig. 2.8bII ) was for incubation and made up of 56 2 × 0.3 mm funnel-shaped microwells (Fig. 2.8c) , with an upper microwell (Fig. 2.8b4 ) and lower microwell (Fig. 2.8b6 ). Paper disks (Fig. 2.8b5) were placed in between the two parts of the microwells. The microwells were created within a few minutes with a simple laser ablation method. This method offers a quick prototyping for developing microfluidic devices by means of high intensity laser beams that evaporate polymers at the focal point. Varying the intensity results in microstructures with different depths. the paper was held in place and prevented backflow of reagents as it is where the antigen or antibody were immobilized. The bottom layer (Fig. 2.8bIII) was fluid removal by means of the outlet channel (Fig. 2. 8b7) leading to a common outlet reservoir (Fig. 2.8b8) . Each channel was connected to a single outlet microwell to act as an outlet reservoir with a negative pressure. For the color change in HBsAg, Fig. 2.8 a Schematic of the hybrid device, b cross-section of the device, c 3DFunnel-shaped microwell, d assembled device with different dyed water, e colorimetric representation of different concentrations of IgG (Sanjay et al. 2016) the antigen was immobilized on the paper surface of the microfluidic microplate, reacting with the primary antibody conjugated with alkaline phosphatase (ALP). The enzymatic reaction between ALP and the colorimetric substrate BCIP/NBT was what produces the purple color. The colorimetric result could be observed by the naked eye within an hour or could be alternatively scanned by an office scanner for quantitative analysis. Figure 2 .8e shows the variety of purple shades that corresponded to the concentrations inserted; the highest IgG concentrations resulted in a darker shade and as that quantity of IgG was decreased, so did the color intensity as is shown from left to right in the image. Li et al. (2018) developed a double-layered microfluidic paper-based device with multiple colorimetric indicators for simultaneous detection of glucose, uric acid, lactate and choline. Linear calibration curves were obtained to identify these biomolecules. These values found from the experiments showed great sensibility (Fig. 2.9a ) by exhibiting very wide linear ranges over two to three orders of magnitude: glucose (0.01-10.0 mmol/L), uric acid (0.01-5.0 mmol/L), lactate (0.04-10.0 mmol/L), and choline (0.04-24.0 mmol/L). The double-layered μPAD was first designed in AutoCAD. Different patterns were needed as the top layer was dedicated to detection and the bottom was auxiliary to construct 3D microfluidic channels. For detection, a 10 mm central sampling zone surrounded by eight 3 × 8 mm microfluidic channels and eight 6 mm detection zones were created. These were modified with colorimetric reagents, different kinds of oxidase and HRP, which can be observed in Fig. 2 .9b. The immobilized chromogenic reagents, once oxidized by the H 2 O 2 from enzymatic reactions between the oxidases and the corresponding substrates, resulted in the color change with co-immobilized HRP as catalyst. The auxiliary layer was made of one 10 mm central sampling zone and eight 3 × 10 mm microfluidic channels connected with eight 6 mm sampling zones. Also, it provided a solution connection by 3D microfluidic channels resulting from overlapping the microfluidic channels and detection zones from the top layer. A traditional waxscreen-printing technique was used to produce the hydrophilic microchannels and hydrophobic barrier on the detection and auxiliary layers. In order to prove the use, a blood sample was introduced into the sampling zone. It was then passed through the hydrophobic channels in order to react with the reactants, thus producing the color, which the Image J software could read. As can also be seen in Fig. 2 .9b, two kinds of colorimetric indicators were used for each biomolecule in order to widen the detection range. This new bilayer microfluidic PAD proved to have a strong colorimetric performance, enhanced sensitivity and extended detection range. Nilghaz et al. (2019) incorporated metal complexation to a μPAD in order to identify antibiotic residues such as oxytetracycline and norfloxacin in pork. This was done by employing the filtration quality of paper combined with aggregation and precipitation of chemical reagents. Ultimately, these processes allowed a LOD and easy result interpretation. For antibiotic residue detection, three layers of filter paper were inserted into a hydrophobic wax paper holder. The topmost layer was made from chromatography paper to serve as the detection zone. In order to detect antibiotic residues, a base substrate made from Whatman #1 and #4 chromatography paper with printed letter channels of both substances from hydrophobic wax paper, was functionalized with copper sulfate pentahydrate in 0.5 M sodium hydroxide and iron nitrate nanohydrate (colorant reagent for oxytetracycline) in a 5 mM ammonia solution (colorant reagent for norfloxacin). A transition metal hydroxide formed when a reaction occurred, allowing the residues to bind to the metal ions through coordination chemistry. In Fig. 2 .10a, a schematic of the individual devices for each antibiotic residue detection can be observed. This complex coupling could result on the filter paper and provided a visible color change as the concentration increased: oxytetracycline was detected with a blue to green color change, while norfloxacin with brown to orange, as can be seen in Fig. 2. 10b. The other two layers were of Whatman #4 filter paper as they were absorbance layers, meant to remove the residual liquid under the base substrate. It is important to note that the colorimetric reagents from the first layer could not diffuse into the bottom layer. The LOD for either was 1 ppm and the recovery rate for oxytetracycline was approximately 88.6% while for norfloxacin recorded to be 111.3%. The whole process of assembly and testing required less than an hour, resulting in a sensitive and rapid method to detect antibiotic residues in food samples. Since the reactions were not interfered by other antibiotics, this device can be implemented to detect other antibiotics from the same families including tetracycline and floxacin. Furthermore, The device has proven to be valuable to food safety surveillance and suitable for large-scale production.

As a common biomolecule for detection, glucose was measured from tear samples in the μPAD biosensor Moreira et al. (Gabriel et al. 2017) designed. The chromogenic reagent used for the samples was 3,3 ,5,5 -tetramethylbenzidine (TMB). The device resulted in a linear behavior between 0.1 and 1.0 mM, as seen in Fig. 2 .11a, analytical sensitivity of 84 AU/mM and LOD of 50 μM. This provides an alternative for diabetic patients pricking their fingers with a lower potential interference, non-invasive, and pain-free sample (Cha et al. 2014) . In order to detect glucose from tears, the desired geometry of the device was designed on Corel DrawTM graphical software and, like other μPADs, was printed on paper substrates by a wax printer (Gabriel et al. 2017) . Effective hydrophobic barriers were fabricated by melting the printer wax while one side of the device was covered with adhesive tape to prevent leaking of the samples. With the basic structure, two 5 mm circular zones, identified as the control and detection zones, and a square region as the sample inlet were defined. The control zone's purpose was to detect potential interferant compounds and to minimize the matrix effect. The sample inlet is evidently where the tears are places to be pulled up to the detection zone by capillary forces. All three zones are connected by a 14 × 2 mm microfluidic channel, whereas the entire device is 24 × 10 mm. The paper (Gabriel et al. 2017) surface was modified with chitosan in order to enhance the surface attachment of enzymes. The chitosan was first prepared in 2% (v/v) acid acetic, subsequently 2 μL of the solution was introduced to the control and detection zones and allowed to dry. The detection zone was spotted with a chromogenic solution of 15 mM of TMB and 120 U mL −1 of an enzymatic mixture of GOx and 30 U mL −1 HRP. The control zone was only spotted with the enzymatic solution. 5 μL of sample aliquots were introduced to the sample inlet and left to reach the detection zone under lateral flow. This can be visualized in Fig. 2.11b . The actual colorimetric detection was done with an office HP scanner with a 600-dpi resolution. Images were taken 15 min after sample addition and were converted to Red-Green-Blue scale for simpler analysis within the Corel Photo-PaintTM software. The color intensity was directly proportional to the concentration of glucose, however, most importantly, it was compared to a personal glucometer, and no statistical difference existed with a confidence level of 95%.

Among different μPADs, Nilghaz et al. (2015) created a compact embeddable microfluidic cloth-based analytical device (μCAD) in order to detect glucose, nitrite and proteins with the naked eye and with concentrations as low as 0.5 mM, 30 μM, and 0.8 mg/dL, respectively. The device proved to be mechanically durable, robust, and flexible (Parikesit 2012) . Cotton was chosen as the raw material for the clothbased analytical device as it is mechanically robust, deliverable to the end user (Nilghaz et al. 2011) , provides an excellent immobilization matrix for biomolecules (Malon et al. 2014) , and a better uniform mixing of reagents and analyte through detection zones (Ballerini et al. 2011; Reches et al. 2010) . Additionally, it can be easily patterned with adhesive wax to create the hydrophobic-wall microfluidic channels. Both wax and cloth are inexpensive Bhandari et al. (2011) and environmentally friendly structural material for disposable diagnostic assays (Park et al. 2004) . Also, cloth-based microfluidic channels can be stable for one week at ambient temperature, making it an optimal factor for application and use in underdeveloped areas (Nilghaz et al. 2015) . Overall, the instrument is a one wax-patterned cloth layer double-inlet device that includes 11 sections among the inlet points, stock zones, detection zones and isolator layers (Nilghaz et al. 2015) . In order to create the 3D colorimetric microfluidic device, the 2D pattern was folded along certain predefined lines. The stock and detection zones were placed in the middle layers and separated by wax-impregnated cloth as isolators. Between 0.1 and 0.5 μL of a solution with colorimetric reagents for glucose, nitrite and protein assays were poured into multiple detection zones by a micropipette, while the detection zone held the reagents for the assay. The traditional wax patterning technique was used to pattern the microfluidic channels on scoured cotton cloth fabric. Furthermore, the ability of wax-patterned cloth fabric with hydrophilic or hydrophobic sections in order to have various designs for multiple bioassays was explored. By stacking layers of individual assay within a small surface area, and separating them by wax-impregnated fabric, multiple assays were able to be conducted. Further improvement was attempted by having an onchip colorimetric calibration by having predefined serially diluted samples next to the detection zones.

Additionally, Lam et al. (2017) developed a chemically patterned μPAD (C-μPAD) by forming hydrophobic barriers using CVD of trichlorosilane (TCS) on chromatography paper. This C-μPAD allowed the measurement of glucose, tumor necrosis factor alpha (TNFA), and heavy metal nickel for point of care diagnostics. To create the structure of the C-μPAD, the desired fluidic pattern was designed in AutoCAD and cut out onto a vinyl tape. This tape was transferred to a 4.5 × 5 cm chromatography paper. In order to silanize the chromatography paper for the hydrophobic barriers, a low-pressure chamber and heat block were required. The vaporized TCS molecules penetrated the paper to bond covalently with hydroxyl groups on cellulose fibers creating an extremely stable and highly reproducible hydrophobic barriers, shown in Fig. 2.12a . The deposition of these TCS molecules depended on pressure, CVD duration, temperature, volume of TCS, and the mobility of the molecules. By controlling these variables, the chemicals traveled through the paper and uniformly immobilized throughout the paper. The patterned paper was placed on a hotplate to remove the vinyl tape to leave the hydrophilic area while other parts remained hydrophobic. This chemically patterned chromatography paper was then evaluated with color dyes, as seen in Fig. 2.12b, c. For glucose, the LOD was 13 mg/dL, which is that of a commercial glucose sensor. The LOD of TNFA was found to be (Lam et al. 2017) 3 ng/dL which again presented similar results as those of the commercial platforms. However, for nickel, a colorimetric agent was immobilized to obtain a stationary and uniform reaction through thermal condensation coupling method. This resulted in the detection of nickel with a LOD as low as 150 μg/dL. These LODs provided high expandability and adaptability for the device. With these results, this C-μPAD produced simple, quick, and cost-effective bioassays for environmental monitoring.

Microfluidic technology has raised an increasing interest in POC diagnostics as it requires small reagent consumption, and offers fast analysis and portability. Capillary and centrifugal forces are the driving forces in these devices that have proven great candidates for integrated genetic analysis due to the versatility of fluidic control without intricate microvalves and tube lines and easy integration of the functional units (Park et al. 2016) . Additionally, expensive and large laboratory set ups may be replaced by smartphones for detection analysis (Wang et al. 2016) . Centrifugal microdevices usually take on the shape of a compact disc (CD) and involve a combination of microfluidic unit operations such as liquid mixing, metering, or valving which are controlled by the rotational speed of the device. Due to this versatility, many applications have forth come such as molecular diagnostics and immunoassay analysis (Sayad et al. 2017 ). Among the developed tests for lab-on-chip platforms, some have found worldwide applications including pregnancy tests (Li et al. 2014 ). Moreover, microfluidic platforms are reported for detection of Tuberculosis (Evans 2017) . Furthermore, new devices are being developed to detect various pathogens for detection of foodborne diseases (Sayad et al. 2017) . Some of the latest examples of the microfluidic BioMEMS used for colorimetric detection are as provided here. Mao et al. (2017) designed a microfluidic chip with eight microchannels in order to determine chlorpyrifos based on peroxidase-like CuFe 2 O 4 /Graphene Quantum Dots magnetic nanoparticles (GQDs MNPs). The nanoparticles were included to amplify the color signal as peroxidase mimetic using a one-step hydrothermal method with electrostatic adsorption. The chlorpyrifos device was made up of a microfluidic chip with an enzyme inhibition reaction, color reaction, and UV spectrophotometric detection areas. The graphene quantum dots were synthesized from a carbonization during the pyrolysis of citric acid. A traditional soft lithography technique was used to create the microfluidic chip where a 50 μm SU-8 photoresist was spun on the silicon wafer. Subsequently, the pattern of the chip was printed on a clear film by 2880 dpi resolution ratio. The male mold of the photoresist was obtained by an ultraviolet exposure for 70 s followed by development. It was mixed with the PDMS prepolymer with a 1:10 ratio and later had air bubbles removed. The mixture was cured for 3 h under 60°C. Later, the inlets and outlets were created on the curing PDMS substance with the microchannel structure by a puncher. Lastly, the PDMS chip was formed by plasma treatment and slide bonding. For testing, 100 μL of chlorpyrifos were injected from the first two entrances to converge at the same point and time. 100 μL of acetylcholine (ACh) and 200 μL NaH 2 PO 4 buffer solutions were added to the second two entrances where the mix flew to the color area. The TMB oxidation produced the color variation and was affected by the H 2 O 2 concentration with the (Mao et al. 2017) CuFe 2 O 4 /GQDs (Fig. 2.13 ). ACh was inhibited as the organophosphate pesticides (OPs) concentration increased, hence reducing the production of H 2 O 2 and thus, provoking a weak color reaction and absorbance. Therefore, it was concluded that the absorbance is inversely proportional to the concentration of OPs.

Another example is a novel biosensor that uses gold nanoparticles to detect the concentration of E. coli O157:H7 equipped with an app for color monitoring (Zheng et al. 2019) . The microfluidic chips have proven to detect foodborne pathogens rapidly due to its precise control of the fluids, few sampling, and decreased detection time.

For the E. coli-detecting biosensor, shown in Fig. 2 .14, the 3D printed, and surface plasma-bonded microfluidic chip was the most important piece. The mold of the chip was placed in 5% NaOH for half an hour and later mixed with curing agent at a ratio of 10:1. It was placed into the mold for 12 h at 65°C and once peeled, it was united with the glass slide through surface plasmon treatment. It had two 600 × 100 μm serpentine mixing channels, where one is to mix the bacterial sample with the MNPs and polystyrene (PS) microspheres, and the other for mixing catalysate with the AuNPs and cross-linking agents. The COMSOL platform was used in these channels to stimulate them based on free triangular grid and finite volume. The chip Fig. 2. 14 Microfluidic colorimetric biosensor for detection of E. coli O157:H7 based on gold nanoparticles and smartphone imaging (Zheng et al. 2019) also incorporated a 14 × 14 × 1 mm chamber that separated the MNP-bacteria-PS complexes and catalyzed hydrogen peroxide. The last part was a 14 × 14 × 2 mm detection chamber where the AuNPs color modifications were observed. The MNPs modified with the capture antibodies and the PSs modified with the detection antibodies were used to interact with the target bacteria in the first mixing channel. AuNP were added for signal indication, Hue-Saturation-Lightness (HSL)-based smartphone imaging correctly detected changes in color, and the microfluidic chip was created for on-chip bioreaction. The results showed that the LOD was 50 CFU/mL for E. coli O157:H7 and the mean recovery was -96.8%.

An analyzer was created by Li et al. for multi-index monitoring of diabetes and hyperlipidemia from a patient's blood Li et al. (2019c) The indexes for monitoring involves glucose (GLU), triglyceride (TG), and total cholesterol (TC). The color changes originated from the peroxidase-H 2 O 2 enzymatic reactions and were taken with a smartphone analyzer that contained a LED light and a charge-coupled device (CCD) camera. The smartphone-assisted microfluidic analyzer contained a 2 mm thick structural layer made from plastic injection molding with pressure sensitive adhesive (PSA) layers on either side. The most important piece was the fan-shaped body of the device with 3.25 cm radius, a buffer pool, four reaction chambers with vent holes, and two positioning holes, each with three capillary stop valves (Fig. 2.15 ). The analyzer was an optical detection system based on step motor, microcontroller, and Bluetooth module. The detection zone included a white LED, macro lens and a CCD camera. To build the microchip, the plastic was first attached to only the bottom layer of adhesive. Subsequently, 13 μL of each detection reagent was input to each of three chambers, leaving one empty to serve as control. The reagents were left there during incubation for 4 h at 37°C and then the top adhesive was added. (Li et al. 2019c) The chip could then be sealed into a vacuum pouch and stored for 6 months at 4°C. In order to test the system, 10 μL of serum was mixed with 190 μL of Tris-HCl and 4-Aminoantipyrine in an Eppendorf tube. 95 μL of the mixture was input to the inlet hole under the pressure of the pipette and the chip went into the Smartphone-assisted microfluidic chemistry analyzer for incubation for 15 min. The detection was done with the phone by means of the light provided by the LED and transmitted by the reagent. It was then collected by the macro lens and hence, detected by the camera as a step motor rotated the chip so all four chambers could be recorded. The images were processed by the microcontroller and sent via Bluetooth to a smartphone for the final analysis. To improve the system, the reagent addition steps could be automated to reduce the manual operation. Additionally, a cost-effective and reliable detector must be put in place to obtain such quantitative results as in Fig. 2 .16. In this device, the detection reagents were mutarotase, glucose oxidase, peroxidase and 2-hydroxy-3,5dichlorobenzenesulfonic acid (DHBS) for glucose; cholesterol esterase, cholesterol oxidase, peroxidase and DHBS for cholesterol; and lipoprotein lipase, glycerokinase, glycerol-3-phosphate oxidase, peroxidase and DHBS for triglyceride.

An Electronics-based ELISA (e-ELISA) using a Lab-on-a-Printed Circuit Board (LoPCB) device for Point of Care (POC) of Tuberculosis was developed by Evans et al. (2017) . The device was a modified ELISA that operated with 10 μL volume and included PMMA wells, gold surface, TMB as the reporter reagent ( Fig. 2.17a ) and Interferon Gamma (IFNγ), a pro-inflammatory cytokine key in innate and acquired immunity, as the assay target. Copper (Cu) foil was laminated to the FR4 PCB substrate through thermal adhesion. Subsequently, tracks made from electrical connections, and electrode pads were patterned by etching the Cu layer. A gold layer was plated on top of the copper one and was fixed by the copper primer which determined the final distribution of gold. The fluid wells were cut from PMMA and fixed onto the surface of the circuit board. The board (Fig. 2.18a ) included reference electrode circuitry, working electrodes with amplification circuitry, voltage input Analogue-to-Digital Converters (ADCs), processing unit and the user interface consisted of an embedded on-board TFT touch screen USB port (Fig. 2.18b) . Both amperometric and colorimetric signals were measured by the device. The first was measured by second generation amperometry where it detected charge carrier concentration through the measurement of total current magnitude charge carriage.

Colorimetric results for each index (Li et al. 2019c) To accomplish this, a reporter molecule was needed that had a relatively low conductivity. For colorimetric readout, capture antibody α IFNγ Fab -3Cys-6His) was immobilized on the Au sensor chip surface and with the addition of cysteine residue. Covalent bonding was achieved, hence capturing and immobilizing antibody fragments by Cysteine (thiol) linkage. The color change was generated from the resulting color of TMB which was originally a colorless liquid that turned bright blue after the reaction (Fig. 2.17b) . The outcome of this study was a lightweight, low-cost, amperometric and colorimetric detection unit made according to standard commercial processes which embedded microfluidics and multi-channel amperometric sensing. 

A digital optical disc (DVD) was used by Li et al. (2014) as the platform for a molecular diagnostic and quantitative pregnancy test. The analytes of interest were human Chorionic Gonadotropin (hCG) identified from urine samples. A standard DVD was prepared for signal readout. The polycarbonate surface was first activated by UV irradiation and then treated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Afterwards, a PDMS plate with six embedded microfluidic channels was placed inside the DVD. Once activated, anti-human Chorionic Gonadotropin Gα (anti-hCGα) monoclonal antibodies were immobilized on the surface (Fig. 2.19a ) by an amide-coupling reaction. A streptavidin nanogold conjugate was added to the surface via biotin-streptavidin interaction following a silver staining treatment, which ultimately resulted in an enhanced signal in order to obtain a significant disruption of the laser readout in the optical drive. A standard, unmodified optical drive was used for the assay readout, and free discquality analysis software for the processing of the obtained data. To use the device, samples were initially loaded into the PDMS microfluidic channels, subsequently, the DVD was spun within the optical drive, undergoing centrifugal forces that created a different radial distance according to the analyte concentration. The assay was tested with a DVD diagnostic software and the readings were interpreted according to the radial distance and optical darkness ratio. The results showed comparable sensitivity and selectivity to well-established colorimetric methods and ELISA (Fig. 2.19b) . Additionally, it is an inexpensive, easy to use, multiplex, POC diagnostic instrument for prompt response used in remote and/or rural areas. Park et al. (2016) developed an integrated rotary microfluidic system (Fig. 2.20a (Park et al. 2016) polymerase chain reaction (PCR), allowing higher specificity and sensitivity, and eliminating the bulky thermocycler. A glass microbead-based centrifugal nucleic acid extraction was used as a solid phase matrix where the genomic DNA could be purified from the lysate sample. Lastly, the colorimetric based lateral flow strip provided a cost-effective and equipment-free detection method. The device integrated these three techniques for detecting in a sequential manner with an optimized microfluidic design and rotational speed control. The microdevice was a five-layer stacked disc with three identical units. Each unit consisted of three functional parts: solid phase DNA extraction, LAMP reaction, and a lateral flow strip. The main three microfluidic layers were created by a CNC milling machine and PSA film was used once the micropattern was cut by a plotter, following a hot press bonding all layers together. The first layer had injection holes and microfluidic channels to transport a LAMP product and a running buffer from the second layer into the lateral flow strip on the third layer. The second layer contained the micropatterns for DNA extraction and amplification. The third layer was for embedding a lateral flow strip for the colorimetric detection ( Fig. 2.20b) . The detection could be made with the naked eye due to the LAMP product. In order to do so, a lateral flow strip containing a buffer loading pad for introducing a running buffer, a conjugate pad (including streptavidin coated AuNPs for the conjugation with the LAMP products), a detection zone (where anti-Digoxigenin, anti-Texas Red and biotin were immobilized in the test line 1, test line 2, and control line, respectively), and an absorbent pad for liquid wicking were incorporated within the device. Thermal guards were patterned around the lateral flow strips to prevent the heat from influencing the anti-haptens on the detection zone of the lateral flow strips. The resulting microdevice presented great potentials as a user-friendly POC analyzer for application in resource-limited setups. The design allowed efficient fluid transfer without sample loss from sample pretreatment to strip detection. The automatic and integrated genetic analysis could then be successfully performed by controlling the rotational speed without the use of expensive equipment.

A cheap and portable smartphone spectrometer for monitoring optical changes as they occur was created by Wang et al. (2016) . The device was aimed at detecting glucose and troponin I, a myocardial infarction biomarker by means of a smartphone with a built-in LED and complementary metal oxide semiconductor (CMOS) camera to use as the light source and the detector, respectively. No external light source, lens, or filter were required. As the dispersive unit, a CD with grating was used. For human cardiac troponin I detection, peptide functionalized AuNPs were taken as the reporters. For the detection of glucose, a solution of 2,2 -azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), HRP, and glucose oxidase (GOx) was utilized. A bi-enzymatic cascade assay was used where the glucose was catalytically converted into hydrogen peroxide, which then converts ABTS by HRP into oxidized form. Once oxidized, a blue color appeared and was read from the color band with the spectrometer bases on the change of intensity. The spectrometer relied on a sample cell with an integrated grating substrate, and the phone's LED flash and camera. The CD was placed 50 mm away from the LED and tilted 5º so that the flashlight passed through a 1 mm diameter pinhole. The grating tracks were aligned to the (Wang 2016) incident light, and the light was refracted from the CD onto the camera (Fig. 2.21 ). This allowed a real-time measurement and resulted in a LOD of 50 ng mL. The biosensing system coupled with a smartphone platform offered a promising method for the phone to detect, interpret, and communicate targeted biological information. Likewise, a higher sensitivity, speed, and simultaneous monitoring was possible. Although it had a comparable performance to commercial devices, it was more compact, cost-effective, and portable. Sayad et al. (2017) created a 165 mm diameter centrifugal microfluidic device platform integrated with LAMP technique (Fig. 2. 22a, c) for quick, monoplex and colorimetric detection of foodborne pathogens. Three main pathogens were studied: Salmonella spp, Es. coli and Vibrio cholerae. 24 strains of these pathogenic bacteria with eight strains of each bacterium were tested and DNA amplification on the microfluidic CD was performed for 60 min. The device consisted of three layers: PMMA top and bottom layers, and a PSA middle layer as shown in Fig. 2. 22d. The layers were aligned and press-bounded together. The top layer contained the venting and loading holes for liquid insertion and wax plug. For the microfluidic structures in the bottom layer, six identical units (Fig. 2.22b) were designed to be able to perform 30 genetic analyses of the three pathogens. One of the units was the loading chamber which loaded the LAMP reagents and primers. Another was the mixing channel and chamber that aliquot the LAMP assay into equal volumes. The sealing chambers contained the sealing material used to seal the connection (Sayad et al. 2017) channel between the metering and amplification chambers to prohibit liquid evaporation. Lastly, the amplification chamber was designed for the amplification and detection of DNA. An optimized square-wave microchannel, metering chambers, and revulsion per minute (RPM) control were utilized to constantly load, mix, and aliquot the LAMP primers/reagents as well as DNA samples. The LAMP reaction amplicons were detected by the calcein dye colorimetric method and analyzed with the developed electronic endpoint detection system (Fig. 2.23a) including the Bluetooth interface to send the results to a smartphone (Fig. 2.23b ). Calcein is a synthetic fluorescein that emits a bright fluorescence creating a visual color change. A positive sample changes from yellow to green while a negative readout remains light orange. The entire process in only one CD lasted about 65 min and presented a LOD of 3 × 10 -5 ng μL −1 . Fig. 2 .23 a Schematic of endpoint detection system, b photograph of endpoint detection system and the application software on a smartphone (Sayad et al. 2017) 

In order to improve the colorimetric biosensing strategy, different amplification methods have been developed. Among these alternative strategies, exonuclease (Exo)-assisted signal amplification, strand displacement amplification (SDA), and rolling circle amplification (RCA) are promising methods. The latter has proven to result in ultrasensitive biosensors due to its excellent properties in signal amplification. However, it creates nonspecific amplification due to the impurity of the circular template, and generation of large fragments of single-stranded DNA (ssDNA) which may decrease the solubility. To counterpart this disadvantage, an improved alternative was developed (Li 2016) . Li et al. (2016) established an RCA-based colorimetric biosensor with an enhanced nucleic acid-based amplification machine to detect attomolar microRNA (miRNA). The machine was composed of a complex of trigger template and cytosine-rich DNA co-modified molecular beacon (MB) and guanine-rich DNA (GDNA) as a probe. This was made by mixing MB and GDNA at a 1.2:1 ratio inclubated for an hour at room temperature. Seal probe was prepared by self-templated ligation of 5phosphorylated dumbbell-shaped DNA sequence using T4 DNA ligase. The machine also required polymerase and nicking enzyme, and a dumbbell-shaped amplification template. The MB template was composed of four sections: miRNA-recognition domain (Fig. 2.24a) , GDNA hybridization domain (Fig. 2.24b) , amplification domain for producing the nickel triggers (Fig. 2.24c) , and a nicking domain for Nb.BbvCl recognition. The target miRNA triggered MB mediated strand displacement to cyclically release nicking triggers, leading to a toehold-initiated RCA (TIRCA) to produce large amounts of GDNAs (Fig. 2.24) . These can stack with hemin to form Gquadruplex/hemin DNAzyme, an HRP mimic, in order to produce a colorimetric reaction. The modified MB decreased the background signal and improved the stringent target recognition. A DYY-6C electrophoresis analyzer was used to perform gel electrophoresis for the seal probe and a Bio-rad ChemDoc XRS for imaging. A NanoDrop 100 spectrophotometer, a UV-visible spectrophotometer, collected the signal. The outcome was a simple, label-free ultrasensitive visual colorimetric biosensor (down to a LOD of 5aM, a detection range of nine orders of magnitude for practical sample analysis). The sensitivity was due to the reduction of steric hindrance and facilitated solution of TIRCA products. The entirety of the process was completed Fig. 2. 24 Schematic of nucleic acid-based amplification machine (Li 2016) within 90 min. The machine itself offered a combination of advantages provided by enzymatic signal amplification and toehold-initiated RCA.

Colorimetric detection can be done visually, hence no expensive equipment or much time is needed. Paper-based devices that operate based on colorimetric detection strategy have improved the accessibility, speed, and accuracy of tests while offering considerable cost effectiveness. Smartphones and tablets opened yet another window of opportunity to easy and onsite analysis of the readout results. By combining microfluidics with the μPADs, the advantages of these devices are as μPAD with colorimetric results attract even more attention due to its simplicity, versatility, straightforward detection results and applicability, especially in point of care analysis without advanced instruments (Li et al. 2018) . Microfluidic colorimetric biosensors offer small size, high precision with small sample size, simple operation, and low cost (Mao et al. 2017) . Overall, colorimetric-based enzymatic assays are fast, adaptable, and cost-effective while allowing the color change to be seen by the naked eye or by digital sensors (Li et al. 2019c) . Centrifugal microfluidic devices present excellent opportunity to detect a variety of biomolecules for different applications. While the literature has witnessed a great deal of advancements in fabrication and application of BioMEMS for colorimetric biosensing, further optimization of these devices for high throughput detection present an opportunity for further improvement.

Gold nanoparticle-based colorimetric biosensors

An inexpensive thread-based system for simple and rapid blood grouping

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Evaluation of commercial glucometer test strips for potential measurement of glucose in tears

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Relevance of neuropeptide Y (NPY) in psychiatry

An assay system for point-of-care diagnosis of tuberculosis using commercially manufactured PCB technology

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Biosensors for epigenetic biomarkers detection: a review

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Multiple semi-quantitative colorimetric assays in compact embeddable microfluidic cloth-based analytical device (μCAD) for effective point-of-care diagnostic

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Textile-based microfluidics: modulated wetting, mixing, sorting, and energy harvesting

An integrated rotary microfluidic system with DNA extraction, loop-mediated isothermal amplification, and lateral flow strip based detection for point-of-care pathogen diagnostics

Biodegradability of cellulose fabrics

Modified biosensor for cholinesterase inhibitors with guinea green b as the color indicator

Thread as a matrix for biomedical assays

Discovering the Beer-Lambert law

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers

Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector

A microdevice for rapid, monoplex and colorimetric detection of foodborne pathogens using a centrifugal microfluidic platform

ELISA-LOC: Lab-on-a-chip for enzyme-linked immunodetection

Multiplex paper-based colorimetric DNA sensor using pyrrolidinyl peptide nucleic acid-induced AgNPs aggregation for detecting MERS-CoV, MTB, and HPV Oligonucleotides

Smartphone spectrometer for colorimetric biosensing

An approach to chemical analysis: its development and practice

Colorimetric sensor arrays for the analysis of beers: a feasibility study

A microfluidic colorimetric biosensor for rapid detection of Escherichia coli O157:H7 using gold nanoparticle aggregation and smart phone imaging