Enzymatic and Enzyme Inhibitory Activity on a Paper-Based Lateral Flow Device

In this work it was demonstrated for the first time that Alkaline Phosphatase (ALP) enzymes spotted and dried onto a nitrocellulose membrane remained active. This was accomplished by passing a fixed volume of the BCIP/NBT substrate solution over the immobilized enzymes and measuring the resulting color density at the spot where the enzymes were spotted. A dose response curve was produced of the concentration of the enzymes spotted on the test area versus the color density measured at the spot after the substrate had flowed past it indicating that the assay produces quantitative as well as qualitative results. This experiment was conducted in paper based lateral flow devices. In a second experiment, prior to the flowing of the substrate over the test spot where ALP enzymes were immobilized, a solution of Sodium Orthovanadate (Na3VO4) was introduced to the system. Sodium Orthovanadate (Na3VO4) is an inhibitor to ALP. By varying the concentration of Na3VO4 in the solution that flowed past the enzymes, a certain number of the enzymes were deactivated. When subsequently the BCIP/NBT substrate solution was flowed over the enzymes, the intensity of the color produced depended on the concentration of Na3VO4. It was then possible to generate again a dose response curve, of the concentration of the Na3VO4 inhibitor in the solution versus the color density at the spot where the enzymes were dried. This is a novel result in that it is again the first time where an inhibitory activity assay was run on a paper based lateral flow device. This experiment also demonstrated that the bond between the Na3VO4 molecules and the immobilized ALP molecules is strong enough so that at least not all of the inhibitor molecules were washed away by the substrate solution.

ix LIST OF FIGURES Figure 1: A: Plasma IaIP levels in newborn sepsis. Blood from newborns with clinically proven sepsis were collected at the time of diagnosis (positive blood culture) and blood from age-matched newborns without evidence of sepsis, were collected as control. B: Septic newborns grouped based on pathogens found in blood culture. [6] .   [4]." From pneumonia to a scraped knee, sepsis can stem from any infection in the human body and is characterized by inflammation as a response to a foreign microbial infection. The most common symptoms for sepsis are fever, chills, rapid breathing, elevated heart rate, confusion, disorientation, muscle and joint pains, a sense of impending doom, rash, and poor feeding (infants and children) [5]. Because these symptoms are unspecific, a diagnosis of sepsis is often delayed [6]. The progression of sepsis in some patients seems to be very slow and they will deteriorate in the late stages of their illness, yet in others, it progresses much more quickly and can be fatal within a few hours [5]. Diagnosis usually relies on blood or tissue cultures that take 6-48 hours. Cultures give false negative results in 30 % of the cases due to antibiotics taken before the test or because sepsis can also be related to toxins produced by the pathogen rather than the pathogen itself [2]. For these reasons, a rapid detection of sepsis is critical to the effort in increasing patient outcomes.

Inter-alpha inhibitor protein and its link to sepsis
Until recent years, the search for biomarkers for sepsis has been largely unsuccessful [2]. In 2000, changes in Inter-alpha Inhibitor Protein (IαIP) levels were linked to inflammation and septic shock, and an IαIP specific enzyme-linked immunosorbent assay (ELISA) was developed [7]. IαIP are plasma-associated serine protease inhibitors that are mainly produced by the liver. Plasma levels in adult sepsis patients are decreased by 20 -90 % and are inversely correlated with unfavorable patient outcomes. In 2009, a decrease in IαIP levels in neonates has also been discovered to indicate bacterial sepsis [6], which can be seen in figure 1. The administration of IαIP to neonatal mice with sepsis has showed improved survival rates [8], reinforcing the link between IαIP and sepsis.

Figure 1:
A: Plasma IaIP levels in newborn sepsis. Blood from newborns with clinically proven sepsis were collected at the time of diagnosis (positive blood culture) and blood from age-matched newborns without evidence of sepsis, were collected as control. B: Septic newborns grouped based on pathogens found in blood culture. [6]

Sandwich ELISA
A sandwich ELISA, specific to each of the heavy chains of IαIP, can quantitatively measure the level of IαIP [7]. A sandwich assay is typically built up on a 96-well micro-titer plate. A 'capture' antibody is immobilized (anchored) on the well surface. Next a sample solution is added to the wells that contains the analyte. The immobilized capture antibodies will bind to the analyte to form the base of the 'assay sandwich.' Next, a buffer solution is used to wash away any unbound proteins, after which a labeled antibody solution is introduced to the assay, completing the 'sandwich' as shown in figure 2. These antibodies are tagged with an enzyme that in the presence of a matched substrate will produce a detectable color change [9].

Competitive ELISA
A sandwich assay is more applicable to high molecular weight analytes such as proteins or allergens. For the detection of low molecular weight analytes, a competitive assay is used. The principle behind this assay is that analytes from the sample must compete with added labelled analytes for immobilized binding sites. The capture antibody specific to the analyte in question is immobilized on the well surface just like in the sandwich assay. Next, a prepared sample solution with the added labeled analytes mixed with the latent sample analytes is introduced. If a high concentration of sample analyte is present, fewer labeled analytes will find binding sites, decreasing signal and vice versa. Figure 3 shows a representation of this competitive assay principle [9].

Point of care devices
Not only for sepsis, but many other diseases progress quickly or can progress unnoticed for a long period of time, thus requiring a quick, cheap, and simple means of diagnosis [10]. Point of Care (POC) devices are an exciting development in medical diagnostics that can be used outside of hospital/laboratory settings and can be used from range of detection applications from diseases and pathogens to explosives and toxins. POC devices are usually one of three main categories: permanent integrated instruments, permanent instruments with disposable components, or pure disposables.
Permanent integrated instruments require the instrument to purge itself of one sample before analyzing another to prevent contamination, and require calibration by a trained technician, which may not be useful in a remote setting [11]. These devices are not typically affordable by individuals and will therefore only useful in labs and hospitals.
Permanent instruments using disposable components have a permanent but portable analyzer that uses disposable cartridges. The analyzer controls the pumping, thermal control, timing, and detection. The disposable cartridges are typically made of glass, silicon, or polydimethylsiloxane (PDMS) and are patterned to contain a microfluidic circuit with reaction and detection chambers where the chemical or biological reactions take place [12] [13]. This prevents the cross contamination from one sample to the next but cartridges can still be rather expensive to manufacture. Pure disposables radically reduce the material and reagent costs of these tests. They are typically run on a paper or other fibrous substrate that can wick fluid through capillary action, removing the need for external pumps. These lateral flow devices (LFD) also reduce the volumes of the reagents needed reducing cost even more. The most noticeable drawback of pure disposable devices is their decreased sensitivity and most cannot produce quantitative results [11].

Paper-based lateral flow devices
Guidelines from the World Health Organization suggest that for the developing world, POC diagnostic devices should follow the acronym ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid, and robust, Equipment-free, and Deliverable to the end-users [12] [14]. Paper-based lateral flow devices (PBD) easily cover the majority of these guidelines, though their sensitivity and specificity are often lacking.
The simplest of such tests are dipsticks such as pH test strips that are treated with a range of concentrations of acid-alkali solutions and will change to certain color depending on the pH of the sample solution being tested [12].

Lateral flow immunosorbent assay
A more complex PBD, a lateral flow immunoassay (LFIA), matches biological reagents to the material properties of the substrate (usually a filter paper or nitrocellulose) to detect various biomarkers in blood, urine, or saliva. As can be seen in figure 4, a LFIA typically has a sample inlet, a conjugate pad, a detection membrane that includes a test line and a control line, and an absorption pad to drive the flow. The main strip of fluid channel is usually made from hydrophilic filter paper to allow the capillary wicking of water-based fluids, while the detection membrane is typically made of porous nitrocellulose or nylon that are tailored to be used with biological reagents.
LFIA commonly use the 'sandwich assay' model for their biological scheme.
After the sample fluid is introduced at the sample inlet, it flows through the conjugate pad, where labeled antibodies conjugate (combine) with the analytes in the sample.
The conjugate then flows farther down the strip passed the test and control lines, where 'capture antibodies' are immobilized and ready to bond with them. The accumulated tagged sandwich complexes can then be detected in many different ways depending on how it was labeled, such as colloidal gold, colored dyes, fluorescent dyes, and magnetic components [15].

LFIA with multiple fluids
For more complex chemistries, more reagents (i.e. more types of fluids) need to be introduced to the LFIA. For enzyme linked immunosorbent assays (ELISA), the labeled antibody is tagged with an enzyme that on its own does not give off any kind of signal. An additional substrate solution needs to be added to the system and when the substrate comes into contact with the enzyme, the enzyme will cleave the substrate. One of the products after conversion will be measurable through a colorimetric or electrochemical signal [13]. The difference between a conventional ELISA test on a micro-titer plate and an ELISA being run on an LFIA can be seen in  where the user can push a button to trigger fluid reservoirs. Figure 6 shows the various paper layers that are need to fabricate the mechanical switch [16]. The requirement for the user to trigger fluids at appropriate times adds the possibility of user error, which is very important to avoid for a diagnostic device. Through the invention of a paperbased fluidic valve [17], complex fluid circuits can now be designed to automatically sequentially load multiple fluid reagents. A representative schematic of a paper-based fluidic valve can be seen in figure 7.

IαIP ELISA on PBD
Once multi-fluid PBDs were possible, Giannakos was able to rapidly measure IαIP levels using the competitive ELISA method. He used a similar version of the three-fluid PBD designed by Gerbers and Foellscher [18] shown in figure 8,

Objective and Motivation
Each additional fluid reagent needed for bio-tests on a PBD adds to its complexity. To detect an enzyme inhibitor concentration quantitatively in a fluid sample, it may be possible to reduce the number of assay fluids to two by utilizing the enzyme inhibitory behavior instead of treating it as a neutral analyte like in the ELISA method. Inter-alpha inhibitory protein (IαIP) blocks the enzymatic activity of trypsin.
Lim uses a 95-well micro-titer plate method to build this assay. First, the trypsin enzyme is added to the unknown sample concentration of IαIP and set to incubate for at least 5 minutes at 37 °C to allow for the trypsin to IαP interaction. Next, the substrate solution, BAPNA, is added and is allowed to develop for precisely 30 minutes at 37 °C. The color of the resulting fluid is then read at 405 nm to measure the change in color [20].
A two-fluid PBD could be designed for this test with a sample inlet and one fluid reagent reservoir. In the nitrocellulose detection spot, a known quantity of the trypsin enzyme is immobilized. The substrate, BAPNA, will start in the reservoir and an unknown concentration of the inhibitor, IαIP, will be in the sample solution.
The enzyme, alkaline phosphatase (ALP), has been used by this lab to label detection antibodies for ELISAs done on a PBD and quantitative results could be obtained. Because ALP's enzymatic activity has already been seen working in a porous nitrocellulose detection area, it was used to model an enzymatic activity test.
Sodium orthovanadate, an inhibitor for ALP, was used to model enzyme inhibitory activity in a two-fluid PBD.

Enzymatic assay for glucose and protein concentration quantification
A group from Harvard University used a multiplexed paper-based lateral flow device, which can be seen in schematic form in figure 9, for a both glucose and protein detection in urine [21]. SU-8 2010 photoresist was used on chromatography paper to pattern the hydrophobic fluid barriers and the hydrophilic paper channels for the devices. The glucose assay they used is based on the oxidation of glucose to gluconic acid and hydrogen peroxide, which is catalyzed by glucose oxidase (notatin). That is immediately followed by the reduction of the hydrogen peroxide and oxidation of iodide to iodine, which is catalyzed by horseradish peroxidase (HRP) [22]. The protein assay they used is based on the nonspecific binding of tetrabromophenol blue (TBPB) to proteins through electrostatic and hydrophobic interactions. The phenol in TBPB deprotonates and changes color from yellow to blue. The brown color of iodine and the blue color from the TBPB were then quantified colorimetrically by the use of a phone, digital camera, or scanner. These images could be sent electronically to an offsite lab to be analyzed or potentially be analyzed by smartphone application. Zhao [23] based a paper-based enzymatic assay on blue colored gold nanoparticle (AuNP) aggregates, modified with S1 and S2 that are cross-linked via DNA hybridization. The cross-linked DNA is cleaved by the enzyme Deoxyribonuclease I (DNase I), which breaks the aggregates. Well dispersed AuNPs appear as a red color. As can be seen in figure 10, the assays were carried out on both hydrophilic and hydrophobic filter paper spots at varying concentrations of DNase I, and photos were taken every 10 seconds.

Alkaline phosphatase
Tests for alkaline phosphatase (ALP) may be used to diagnose liver or bone disease. Increased ALP levels can also be seen in children undergoing growth spurts and in pregnant women. Normal ALP levels in blood from adults are 44 to 147 IU/L [24]. ALP is also found in neutral or alkaline soils, while acid phosphatase (AcdP) is found in acidic soils. An appropriate pH for crop growth can then be defined as a soil with a proper AcdP/ALP activity ratio [25].
The substrate for ALP to be used in the enzyme activity tests needs to convert on the same time scale as the fluid flowing passed the detection spot, which typically takes less than 10 minutes depending on the fluid volumes and channel geometries.
NBT/BCIP was chosen as color development happens in ~5 minutes [26]. Another advantage of this substrate is that the product after conversion is a dark purple precipitate that can be easily seen on a white nitrocellulose background. The fact that it precipitates allows the color to accumulate in the porous nitrocellulose, because the particles are too large for unhindered flow [27].
Peng [28] used a spot test on filter paper to test ALP activity with the NBT/BCIP substrate. ALP was immobilized on filter paper using a vacuum method.
Three microliters of various dilutions of BCIP/NBT substrate solution were added to the spot and left to convert for 30 minutes. It was scanned at 600 dpi and converted from RGB brightness values to a mean gray brightness value (V) using the formula (V=0.299R+0.587G+0.114B), which are the default weighting factors used to convert RGB to YUV, the color encoding system used for analog television [29]. Ideally, weighting factors to match the dark purple color produced by this substrate conversion should be used. Peng's data can be visualized in figure 11. A group from Monash University, in Austrailia, patterned a multiplexed PBD using alkyl ketene dimer (AKD) to hydrophobize filter paper and a plasma treatment through a metal patterning mask to rehydrophilize the regions designated to be fluid channels [30]. The ALP was divided into two parts; one was heated for 10 minutes at >70 °C to deactivate the enzyme. They spotted the active and deactivated ALP into designated filter paper tests spots at the ends of fluid channels. As can be seen in figure 12, after the substrate, BCIP/NBT, was added at the intersection of the six channels and allowed to wick for a short time, test spots with active ALP enzymes changed to a purple color, whereas test spots with the deactivated ALP did not.

Figure 12:
Three active and three deactivated ALP enzyme sample reacting with the liquid substrate BCIP/NBT [30].
Giannakos [19] immobilized various concentrations of mouse monoclonal and polyclonal antibodies tagged with ALP onto a nitrocellulose detection area in a PBD.
He also used the BCIP/NBT substrate but in a lateral flow format rather than the direct spotting and incubation. The chips were scanned and analyzed the same way as Peng's in method [28], though only single data points were gathered for each concentration, leaving a lot of uncertainty. Figure 13 shows the data gathered with error bars equal to plus and minus two standard deviations of the individual pixel brightness values that were averaged together to form each data point. These error bars were calculated incorrectly, as they should be two times the standard deviation of multiple RGB mean gray value data points, should the same 95 % confidence level scheme be followed.

Inhibitory activity of sodium orthovanadate on ALP
Seargeant found that sodium orthovanadate can be used to inhibit the enzymatic activity of ALP produced by a human liver, small intestine, or kidney. The inhibition of ALP activity by orthovanadate can be seen in figure 14. The specific activity of the enzyme from liver was 1300 U/mg [31]. One unit of enzymatic activity, U, is equal to the amount of enzyme that decomposes 1 µmole of substrate per minute at room temperature [32].

Paper-based fluidic valve
What makes multi-fluid PBDs possible is a single-use paper-based fluidic valve developed by Dr. Hong Chen. He used the chemistry of surfactants to allow water based fluids to wick through hydrophobic paper. Surfactants, like the tween20 used in these valves, is a molecule that has both a hydrophilic head and a hydrophobic tail.
Surfactants are surface active molecules that, when mixed with water, will poke their hydrophobic tails through the water-air interface, as seen in figure 15.   Chen was also able to develop an improved version of the one-way fluidic valve that is built from multiple layers of filter paper and double sided tape. As can be seen in figure 17, hydrophilic filter paper channels are lined up to intersect over a hole in water impermeable double sided tape. A hydrophobic filter paper disc is set in the hole to block fluid flow and maintain contact between the two filter paper layers through the hole. Surfactant is spotted on one of the filter paper sides to allow fluid flow to pass the hydrophobic filter paper disc only from that side. This basic 3-d oneway fluid valve became a major building block in the development of complex paper-based fluidic circuits that could then be used for biomarker detection using a variety of multi-fluid assays.

Methodology
In this chapter, the methodology for the fabrication of the paper-based lateral flow devices will be explained in detail. This includes a list of the materials needed, the required tools and equipment, the fabrication of individual parts, the process to assemble the parts, and how to prepare the reagents and experiments for enzyme activity and enzyme inhibitor activity tests. Small tweaks to the conventional fabrication process are also discussed.

Chip Fabrication Introduction
What makes the paper-based lateral flow devices designed by this research group unique is the ability to automatically handle multiple fluid reagents and sequentially introduce them to the assay. Wax is printed onto filter paper and then melted to produce hydrophobic walls impenetrable by water based fluids. The patterning of these walls produces fluid channels and other fluid manipulation structures like paper level changes and 3d fluidic valves. Important parameters to consider when designing such a chip are channel geometries (length and width), valve sizes, filter paper porosity and dimensions, and wax melting times.
A typical chip design used by this research group is one designed by Gerbers and Föllscher [18]. Despite the fact that in the meantime there have been improvements to this design, it is still a good representation of chips currently being used. The top layer contains a sample inlet, two reservoirs, a potential conjugate pad, a detection zone made of nitrocellulose, and a large connection to the waste pad. The second layer is a connection layer that separates the top layer from the timing channels. In this design it is made from double sided tape, filter paper discs treated to by hydrophobic, filter paper discs that contain the surfactant tween20, and some native filter paper discs, as well as a large piece of glass fiber paper to help absorb and transfer waste fluid to the absorption pad. This layer is improved in the work by Alex Giannakos [19]. He replaces the double sided tape with 3M ® Super 77 Multipurpose Adhesive and consolidates the hydrophobic and surfactant discs into the filter paper layers. This improvement will be seen in the fabrication of the protocol development

Paper-based chip fabrication
Materials, software, and equipment needed for paper chip device fabrication:

CO2 laser cutter settings
Using the same drawing software, cut-files are designed to be used with the Epilog ® Mini 24 CO 2 laser cutter. This allows for multiple chips to be cut from larger sheets of filter paper, scaling up the fabrication speed. For the design shown in figure  18, six paper-based later flow devices are printed per chip and six chips can be printed on a single 8 x 10 inch (203 x 254 mm) piece of filter paper. Along with any holes that need to be cut into the filter paper layers or double sided tape, the edges of the chips need to be precisely cut to dimension so that they can be fit into alignment fixtures used during assembly. Not only the filter paper layers but the double sided tape, glass fiber paper, nitrocellulose pieces, conjugate pads, and blotting paper layer need to be laser cut. As can be seen in Cut files are designed as vector files in such a way that the laser can follow a path to cut. To align the designed cut file to the wax printed structures on the filter paper, alignment guide markers are used. For the initial rough placement of the filter paper, two orthogonal black wax lines (can be seen in figure 19) that are printed along with the wax patterns is aligned with the edge rulers of the laser cutter. Using the preprogrammed filter paper cut align laser cutter settings, hairline width crosses are cut over alignment guides also seen in figure 19. The main purpose of the alignment lines is to place the wax printed filter paper parallel to the cut file. The alignment guides can then be used to shift the cut file horizontally, in the x-direction, and vertically, in the y-direction, to precisely match the placed wax printed filter paper.
There are two alignment guides per sheet. Should both alignment guides show differing offsets, it means the wax printed filter paper is not placed with the wax alignment lines parallel with the ruler edges of the laser cutter and needs to be adjusted.

Alignment lines
Alignment guides Figure 19: Cutting guides [19] To cut the waste/absorption pad pieces, the blotting paper cut file is used. The Whatman Grade GB003 blotting paper is simply aligned along the laser cutter ruler edges and do not need additional alignment steps as no wax structures are printed on it. As blotting paper is thicker, it needs additional power to be cut. The power, frequency, and speed settings from table 2 are used.
The  It is important to note that the printed wax structures will intrude laterally up to 0.5 mm into the native paper regions during melting and need to be compensated for in the initial design [19]. This expansion also happens to the wax printed alignment markers used during laser cutting, which makes it important to laser cut the chips to size before melting as the markers will be far less precise post melting.
Before melting After melting

Chip assembly
Before assembly, designated spots on the hydrophobic layer need to be treated with a solution of allytrichlorosilane. This treatment makes these areas chemically hydrophobic but do not fill the filter paper pores like the wax. Surfactant is also deposited in assigned locations on the second filter paper layer. The combination of the chemically hydrophobic spots and the surfactant produce the fluidic valve described in section 2.3. After all the layers and materials have been prepared, the chip can be assembled. The edge of each layer was cut to fit a red alignment tool that can be seen in figure 22. The layers are combined, starting by inserting the blotting paper layer into the alignment tool. The third tape layer, which separates the fluid channels from the second filter paper layer from the absorption pad, is aligned and pressed firmly onto the blotting paper in the alignment tool. The second filter paper layer is placed on top of that and is followed by the second tape layer and then the hydrophobic layer. The pieces of nitrocellulose and the conjugate pads are then precisely placed on designated locations on the hydrophobic layer. The first tape layer is then aligned and placed over the hydrophobic layer, securing the nitrocellulose pieces and conjugate pads. The first filter paper layer is then placed onto the first tape layer followed by the top clear tape layer, which is there to prevent evaporation and contamination from and to the chip.

Protocol development chip
A protocol development chip was developed as a way to focus on a biological test and its reagent interactions that only uses two fluids instead of three. As can be seen in figure 23, its functional components are filter paper channels, a single fluidic valve, a nitrocellulose detection area, and a blotting paper absorption pad. It is fabricated the same way as the three-fluid chip but requires only a single spot to by chemically treated with allytrichlorosilane to be hydrophobic. Because of the smaller size, eight paper based devices fit on each chip, two more than with the larger threefluid design.

Spray adhesive method
A student from this research group, Benedikt Beermann, replaced the tape layers with a spray adhesive. This reduces the chip complexity and shortens the fabrication time by up to 80 % [37]. Figures 24 and 25 show the modifications made to the wax patterns on the filter paper layers. The surfactant is now directly spotted onto the fourth filter paper layer and a hydrophobic disc is used in place of the hydrophobic layer. To assemble the layers, the same procedure is used with the alignment tool except for the fact that the layers need to be sprayed with the adhesive before being attached to chip in the alignment tool. The adhesive is sprayed in two passes from approximately 1-2 feet away and as evenly across the chips as possible.  For the enzyme activity and enzyme inhibitor activity tests the chip was simplified even further. The second and third layers were removed from the design, essentially removing the reservoir and paper based fluidic valve from the chip, and turning it into a conventional strip test. This change was done to focus the research on the biological and chemical aspects of the chips. Lacking the automatic sequential loading of fluids to the chip, reagents for this simpler protocol development chip needed to be added to the sample inlet by hand. This avoids the need to optimize channel geometries for timing channels and fluidic paper valves, removes the chance of unknown effects from the surfactant, tween20, on the biological reactions, and allows for variations in incubation times without having to optimize a new chip design. These simplified chips were also ideal for testing enzyme activity as it made it easy to vary reagent volumes without having to change the chip design.

Alkaline phosphatase activity
The enzyme, alkaline phosphatase (ALP), was used as an initial benchmark to measure enzyme activity with a paper based lateral flow device. It was chosen as it quickly converts the substrate, BCIP/NBT, into a dark purple precipitate that is highly visible when present in white nitrocellulose. Depending on the pore size of the nitrocellulose, the precipitate will also be physically hindered from flowing downstream in the chip.

Materials, equipment, and reagents needed for ALP activity test:
 Simplified single-fluid protocol development chips After the substrate has finished flowing, the chip is allowed to dry for an hour and is then scanned in color at 600 DPI. Figure 26 shows an example scan of a single chip with seven tests (plus one background at the bottom right). The image is opened using the ImageJ software and the colors are inverted. In ImageJ and using a Red, Green, Blue (RGB) color scheme, the signal is measured per pixel as a brightness value in the range from 0 to 255. A circle is drawn around the detection spot (yellow circle in

Sodium orthovanadate inhibitory activity
In this section sodium orthovanadate (Na 3 VO 4 ) is used in solution form to inhibit the enzyme activity. This is to test how varying concentrations of an inhibitor in a sample solution will block the enzyme from converting substrate.
To make the sodium orthovanadate solution, the following steps need to be followed: As the substrate solution passes the nitrocellulose detection spot, a purple color may be seen depending on the concentration of inhibitor used on that chip. The chips are then allowed to dry for one hour before being scanned in to collect the quantitative data.
The same scanning procedure is used as with the ALP activity experiments in the previous section. The chips are scanned in color at 600 DPI, their colors are inverted using ImageJ, and a mean gray value is measured from a circular area chosen in the detection spot. The difference for these chips though is that a high signal means low ALP enzyme activity and thus a high Na 3 VO 4 inhibitory activity, while a low signal means high ALP activity and thus a low Na 3 VO 4 activity.

ALP substrate volume test
For this experiment, the effect of the substrate volume was investigated. The approximately 20 µL of fluid for the substrate to even reach the nitrocellulose detection spot. Therefore, when looking at the data, signal will only start appearing with volumes higher than 20 µL for all tests. After the chips were dried following the same procedure as the other ALP enzyme activity tests, the chips were scanned and the data analyzed using ImageJ.

Trypsin activity
The next tests were done as a precursor to the full-fledged sepsis test. For sepsis, the concentration of the enzyme inhibitor, inter alpha inhibitor protein (IαIP), is measured. This inhibitor blocks the enzyme activity of trypsin from converting the substrate, BAPNA. Before testing the activity of the inhibitor, the enzyme activity of trypsin first needs to be tested.
The materials, equipment, and reagents needed for trypsin enzyme activity tests:  scanned. Using ImageJ, the chips are analyzed using the same procedure as in the previous test, section 3.5, and using the same RGB mean gray value method and color weighting for an inverted yellow to blue signal. The same test was done again using only the highest trypsin concentration available and increasing the substrate volume to 400 µL, pipetting the substrate 20 µL at a time to avoid the fluid from overflowing.

Findings
The first thing that needed to be tested was to see whether a basic enzymatic activity test could be performed using a paper-based lateral flow device. To prove that such a test is possible, a well-known enzyme, alkaline phosphatase (ALP), with respective substrate, BCIP/NBT, and inhibitor, sodium orthovanadate, combination was selected to be tested using a simplified version of the protocol development chip fabricated in-house. This simplified chip was developed as a way to focus the research on the biological test and its behavior in a porous substrate, which in this case is nitrocellulose. Using this chip avoided interactions with other aspects of the chip such as the surfactant and chemicals, which are used for paper sizing, present in the paperbased fluidic valves. As there was only the one single fluid channel in this design, the timing between the introductions of fluid reagents as well as incubation times were all done manually. This is advantageous when testing new unproven biological tests as the incubation times still need to be optimized and can then be easily adjusted. Should the original protocol development chip be used for such tests, the chip design would have to be altered every time the timing of the fluid reagents needs to be changed by, for example, changing the fluid channel lengths.

Alkaline phosphatase activity
To model different levels of enzymatic activity of alkaline phosphatase (ALP), concentrations of 0, 5e -4 , 1e -3 , 5e -3 , 1e -2 , 5e -2 , and 1e -1 mg/mL of ALP in 1X PBS were spotted and allowed to dry on the nitrocellulose detection spots. After the drying and  Each chip contains eight paper based lateral flow devices, seven of which are had the spotted ALP and the eighth was used to measure the background signal as no ALP was spotted there. The mean gray values of the seven tests of each concentration were averaged together and the standard deviation calculated. For the error bars that will be used to graph the data, a 95 % confidence interval was used, which equates to two times the standard deviation, plus and minus the average of the data points. The average of these data points can be seen in

Alkaline Phosphatase (ALP) Activity
The stock ALP solution was acquired having a concentration of 5 mg/mL. The reason that the highest concentration shown in the data set in table 2 and figure 29 is 1e -1 mg/mL, is that above those ALP concentrations the chip becomes oversaturated.
At such high enzyme concentrations, the ALP converts the substrate far too quickly and the resulting precipitate clogs the porous substrate. This clogging forces the remaining substrate fluid to wick around the sides of the detection area, where no enzyme is present, and thus will not be converted. Figure 31 shows

Sodium orthovanadate inhibitory activity
The next step was to discover the effect of an inhibitor, in this case sodium  In figure 32, the tabulated numbers can be visually compared and it is easily recognizable that with an increased concentration of inhibitor the measured mean gray value decreases as expected. At inhibitor concentrations below the 1:1e 3 dilution, the range in the error bars start to blend together. At the highest inhibitor concentration the measured mean gray value sinks below the measured mean gray value of the background tests. It should also be pointed out that with an increase in inhibitor concentration, the size of the error bars also increase. Possible solutions to these phenomena will be discussed in chapter 5. Enzyme Inhibitory Activity

ALP substrate volume test
Because it is the substrate that is converted into the detectable signal, and because there is a wide range of volumes that potentially could be used for these devices, an appropriate volume needed to be found for such tests.

Enzymatic and inhibitory activity of trypsins
Inter-alpha inhibitor protein (IαIP), an inhibitor for the enzyme, trypsin, and a biomarker for sepsis, is the next analyte of interest to be detected using an enzyme inhibitor activity assay. Because these reagents behave similarly to the ALP/sodium orthovanadate assay, the tests using trypsin and IαIP were developed the same way.

Trypsin activity
Before the detection of the inhibitor can be done, the enzymatic activity of the trypsin needed to be tested. Various concentration of the trypsin solution were spotted and immobilized on the nitrocellulose detection area and after it was allowed to dry, covered by a piece of transparent tape. A 40 µL volume of the substrate, BAPNA, was then introduced to the fluid inlet and allowed to wick through the paper-based lateral flow device and over the nitrocellulose detection area where the trypsin was immobilized. As the fluid crossed the detection area there was no visible change in color.

Trypsin activity with increased substrate volume
The same test was repeated with some changes in an effort to obtain even a

Inter-alpha inhibitory protein activity
For this test it is necessary to have a control and high starting signal as the role of the inhibitor in this assay is to decrease the enzymatic activity, which in turn reduces the resulting signal. Because the previous test (section 4.4.2) did not produce high enough signal, the experiments for this section could not be carried out.

Conclusion and Future Work
The research done into enzymatic activity and enzyme inhibitory activity assays on paper-based lateral flow devices progressed in several ways over the course of this research. The most important of these was the proof of concept that enzymatic activity and enzyme inhibitory activity tests are possible on a paper-based lateral flow device. The conventional method for an enzymatic activity and enzyme inhibitory activity tests are run using elaborate and expensive methods that require large amounts of sample fluids and reagents, expensive equipment, a laboratory, and a trained technician [20]. This new method potentially allows for the elimination of all of these requirements with some additional optimization and product design work.

ALP enzyme and inhibitory assay
For the alkaline phosphatase (ALP) enzyme, data was acquired showing the possibility of a signal to enzyme concentration calibration curve (figure 29 in section 4.1). The very rough method of spotting the enzyme, however, causes rather large ranges in measured values for tests with identical parameter sets. Once that has been optimized, a calibration curve useful for marketable products will be possible. For the enzyme inhibitory activity of sodium orthovanadate, using the ALP assay, the same conclusions can be made. A preliminary signal to inhibitor concentration curve ( figure   32 in section 4.2) was achieved, yet the error range of this data also leaves much room for improvement through optimization and other engineering methods. This curve also proves that the binding of the inhibitor to the enzyme is strong enough and will not be broken as the substrate solution flows by.
The shape of the spot that changes color is far smaller than the area that gets wetted when spotting the ALP enzyme solution, which indicates that the ALP molecules quickly immobilize on the nitrocellulose during the spotting. The volume and concentration of the enzyme spotted both have an effect on the final size and shape of the immobilized enzyme and cannot be measured or controlled before the tests are carried out. A new method for the deposition of the enzyme reagent needs to be found. Using an inkjet printer has proved promising to precisely deposit small volumes of biological liquid reagents [39]. Printing the enzyme as a test line instead of a spot will not only solve the problem of controlling the size, shape, and concentration of the immobilized enzyme detection area, but also the problems with uneven brightness values measured and the wicking of substrate fluid around the sides of the detection spot. Many of these problems are due to local areas of a highly concentrated substrate precipitate particles clogging the small pores in the nitrocellulose. If the enzymes were printed as a test line similar to conventional lateral flow tests, the substrate and other reagents will not be able to bypass the functionalized detection spot, preventing the loss of a large portion of the signal strength.
To improve these tests even further, a washing step can be introduced to the chip design. After the substrate solution is finished flowing, any unconverted substrate could be washed away with an additional fluid, because as the fluid dries, any unconverted substrate may cleave to give falls signal both in the detection area and on the background. Increasing the color brightness of a background measurement will have a huge effect on the signal to noise ratio as the noise is calculated from the background brightness value. Also another enzyme, horseradish peroxidase (HRP) could be used along with an appropriate precipitating substrate like 3,3'-Diaminobenzidine (DAB), to perform similar enzymatic activity tests. As a potential application, the inhibitory activity of cyanide on the HRP enzyme could be quantitatively be measure with a similar PBD assay as the ALP enzyme inhibitory activity assay. HRP also has the advantage that it is also useable at a wider range of pH levels [40].

Trypsin activity
The enzymatic activity tests done using trypsin had far less promising results.
The chemistry involved in this test does not fit well with paper-based lateral flow devices. There are several problems that need to be addressed. The first, the time required for the enzyme, trypsin, to convert the substrate, BAPNA, into its colored product, from which one receives the signal, take much longer than in the ALP activity tests. The substrate takes over 30 minutes to convert in the conventional 96well microplate method, while it takes less than 3 minutes for the same volume of substrate solution to flow through the paper-based lateral flow device. Another issue is that the colored product is not a precipitate like in the ALP assay, but a soluble yellow dye. The advantage of a colored product that is a precipitate is that it will stay stuck between the pores of the nitrocellulose detection spot producing small areas of high concentration that can be easily detected. The soluble dye, however, will continuously be washed into the waste/absorption pad as fresh substrate solution enters the detection area. The yellow color of the dye produced in the trypsin/BAPNA assay is also difficult to see and measure on the white nitrocellulose background. The simplest solution would be to find an alternative substrate that when converted by trypsin, turns a darker color. Hide-Remazol Brillian Blue R from SigmaAldrich ® may be a substrate worth investigating for future tests. This substrate is converted from a blue color to orange (595nm), colors that contrast each other strongly and can easily be detected on the white nitrocellulose background. Ideally, a substrate that also precipitates when converted should be chosen. It is unclear at this point whether this substrate is a precipitate before or after it is converted. Eventually the paper-based lateral flow devices with the trypsin/IαIP assay should produce results comparable to the conventional 96-well microplate method like in figure 36, which shows the optical density with respect to the inhibitory activity of the IαIP. After the reagent concentrations, volumes, and incubation times for these assays are optimized, a full 3d paper-based lateral flow device can then be designed with appropriate timing channels to account for the incubation times, and paper-based 3d fluidic valves to trigger and sequentially load various fluidic reagents. The assays done in this work all require a minimum of two fluids when they are run, the sample fluid containing the inhibitor solution (or a wash if only and enzyme activity test) and the substrate in a separate reservoir. The order and timing at which each of them enters the detection zone are automatically controlled by the chip. A protocol development chip, a two fluid design, as can be seen in figure 37, was designed to handle two fluids in a sequential loading scheme [19]. The sample fluid holding an unknown concentration of the enzyme inhibitor is added at the sample inlet. A defined volume of substrate will be added at the reservoir inlet and its flow will be stopped due to a hydrophobic fluidic valve in the layer beneath it. Once the sample fluid reaches the other side of the fluidic valve, it mixes with a surfactant dried in the paper and opens the valve, opening a path for the substrate solution to flow through the detection spot.
The design would have to be optimized to account for incubation times but would roughly function as is. Figure 37: Design of two-fluid protocol development chip [19].