Simple, Cost-Effective 3D Printed Microfluidic ... - ACS Publications

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Simple, Cost-Effective 3D Printed Microfluidic Components for Disposable, Point-of-Care Colorimetric Analysis Ho Nam Chan,† Yiwei Shu,† Bin Xiong,† Yangfan Chen,† Yin Chen,‡ Qian Tian,†,‡ Sean A. Michael,† Bo Shen,† and Hongkai Wu*,†,‡ †

Department of Chemistry and ‡Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China S Supporting Information *

ABSTRACT: The fabrication of microfluidic chips can be simplified and accelerated by three-dimensional (3D) printing. However, all of the current designs of 3D printed microchips require off-chip bulky equipment to operate, which hindered their applications in the point-of-care (POC) setting. In this work, we demonstrate a new class of movable 3D printed microfluidic chip components, including torque-actuated pump and valve, rotary valve, and pushing valve, which can be operated manually without any off-chip bulky equipment such as syringe pump and gas pressure source. By integrating these components, we developed a user-friendly 3D printed chip that can perform general colorimetric assays. Protein quantification was performed on artificial urine samples as a proof-of-concept model with a smartphone used as the imaging platform. The protein was quantified linearly and was within the physiologically relevant range for humans. We believe that the demonstrated components and designs can expand the functionalities and potential applications of 3D printed microfluidic chip and thus provoke more investigation on manufacturing lab-on-a-chip devices by 3D printers. KEYWORDS: 3D Printing, point-of-care device, smartphone-based quantification, colorimetric analysis, microfluidics, microfluidic valves

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gradient generation, and detection of chemicals such as glucose and nitrate were demonstrated.14,15 However, in the context of point-of-care (POC) analysis, where simplicity and cost are critical,16 most of these developed designs cannot be applied. This is because they require the bulky off-chip equipment such as pumps, gas pressure sources, pressure regulators, and microscopes for operation and detection. This significantly offsets the advantages brought by miniaturization.16,17 Developing bulk equipment free components for 3D printed microfluidic device control can facilitate the development of the “lab-on-a-chip” (but not “chip-in-a-lab”) devices. Apart from 3D printed microfluidics, paper-based microfluidics is another promising technology in POC analysis because of its low manufacturing cost and simple operating protocol. Currently, many groups are actively adding more controlling elements to enrich the functions of paper-based devices.18−20 However, paper-based devices have their own challenges and practical limitations in providing high quality analytical results. For example, in colorimetric analysis, because of the opacity of paper (i.e., short optical path), colored

hree-dimensional (3D) printing is an emerging technology that builds 3D objects easily and quickly. With the improvement of printer resolution and decreasing price, 3D printing has become an attractive method for the fabrication of microfluidic chips. Because it is an additive manufacturing process, 3D printing-based production effectiveness is inherently higher than traditional subtractive manufacturing methods.1,2 Also, without modifying hardware, the same set of printers can produce different microchip designs, which endows 3D printing with a great potential for mass production and customization of microfluidic chips. However, previously developed polydimethylsiloxane (PDMS) based microfluidic components, which are essential in expanding chip functionalities, such as valves3−5 and on-chip micropumps,3,6 cannot be directly applied on the 3D printed microchips because of the incompatibility of chip material. Recently, some components and applications tailored for 3D printed microfluidic chips have been developed. For example, 3D printed chips integrated with electrodes and cell culture inserts were developed for cell study;7−9 3D printed microfluidic reactionware was investigated for direct chemical synthesis;10,11 luer interface and gas-pressure driven peristaltic valves and pumps were introduced to detect pathogenic bacteria2,12 and manipulate fluid on chip;13 separation, mixing, © 2015 American Chemical Society

Received: September 10, 2015 Accepted: December 11, 2015 Published: December 11, 2015 227

DOI: 10.1021/acssensors.5b00100 ACS Sens. 2016, 1, 227−234

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assay reactant was diluted to 1/2.5 (for low concentrated protein detection chamber) and 1/5.5 (for medium and high concentrated protein detection chamber) of the original concentrate by distilled water prior to loading in the reaction chamber. Sample Preparation. Artificial urine spiked with bovine serum albumin (BSA, Sigma-Aldrich, USA) was used as the model sample solution. The artificial urine was prepared as previously described.23 It contained 1.1 mM lactic acid, 2.0 mM citric acid, 25 mM sodium bicarbonate, 170 mM urea, 2.5 mM calcium chloride, 90 mM sodium chloride, 2.0 mM magnesium sulfate, 10 mM sodium sulfate, 7.0 mM potassium dihydrogen phosphate, 7.0 mM dipotassium hydrogen phosphate, and 25 mM ammonium chloride. The final pH was adjusted to 6.0 by addition of hydrochloric acid. BSA was used to represent the protein sample since albumin is the most abundant urinary protein in patients with proteinuria.24 Smartphone-Based Quantification of Urinary Protein. Diagnostic technology typically requires imaging to quantify the amount of analyte present. The smartphone is a good platform for this due to its built in imaging, processing and communication capability, and popularity.25,26 However, current smartphones are not designed for precise analytical application. We have developed some accessories that can be used to transform this device into an image quantification instrument.27 Here, a simple prototype of the smartphone accessory was built for urinary protein quantification. It consisted of two parts: (1) core setup and (2) light shield (Figure S-2a,b). The assembled core setup is shown in Figure S-2c. At the bottom of the core setup, there is an electronic circuit, which connects the 595 nm LED to a battery, resistor, and on/off switch. The 595 nm LED was used to fit the absorption maximum (λmax) of the reacted Coomasie Brilliant blue G250 dye (595 nm) in order to enhance the quantification quality. 3.5 cm above the LED (a point light source), a diffuser (an A4 paper sheet) was installed to gain uniform illumination of the chip. A chip holder was located above the diffuser, which allowed users to insert the incubated chip from the side for image acquisition. Because a smartphone camera was not designed to take high-quality macro images, an optical lens (plano-convex, Φ = 25.4 mm, f = 40 mm, ChangchunFortuneOptronics, Inc., China) was installed to aid focusing. The entire setup functioned as a simplified optical spectrometer. The LED was chosen to match the wavelength of absorption peak, which reduced interference and enhanced detection sensitivity. To eliminate the interference caused by surrounding light, a custom light shield was used to cover the setup and also served as a support for the smartphone during image acquisition (Figure S-2d,e). Image Acquisition and Processing. An iPhone 5S (with an 8.0 megapixel camera) was used as the model smartphone in this work. The images were taken by the native camera application (iOS 7.1.1) and sent to a computer for further processing. As the light source was chosen to match the absorption maximum of the reacted assay reagent, only the information on light intensity in the region of interest was required to obtain the absorbance. Thus, the acquired colored image was converted to 8-bit gray scale format to combine the raw three RGB (Red, Green, and Blue) channel data into a single gray channel light intensity data. After extracting the light intensity data by ImageJ, absorbance was calculated with the formula Abs = −log(I1/Io), where I1 is the average light intensity of the reaction chamber and Io is that of the reference region (see Figure S-3 for the location of the regions). As demonstrated in the literature, dedicated smartphone applications can be written in practical situations to leverage the computing power of smartphones and thus eliminate the needs of an external computer for image analysis.27−30

reporting dye has to be concentrated in order to be detected, which decreases the sensitivity of the assay. 21 Also, inhomogeneity of color distribution can hinder the quantification process.22 Furthermore, the environmental temperature and moisture can affect the capillary action of fluid which may reduce the reproducibility of paper-based devices.21 In contrast, 3D printed microfluidic device does not suffer from these problems. Optical path can be adjusted by the height of the channel, and the sample can be actively loaded into the device. In this work, we report a new class of movable chip components tailored for 3D printed chip, including simple pump and various types of valves, that can be operated manually. These components are developed to enrich the fluidic manipulation capability of 3D printed chips without the needs of bulky off-chip equipment. Furthermore, we integrated the torque-actuated pump and pushing valves on a 3D printed chip to demonstrate a POC application for urinary protein quantification. The procedure consists of three steps: (1) introduce the sample onto the injection port, (2) rotate the torque-actuated pump, and (3) push the valve using a finger to run the protein assay. The assay results were quantified via a smartphone-based detection platform with minimal accessories; and the whole assay was done within 25 min. Furthermore, by incorporating appropriate assay reagents, the developed 3D printed chip can be applied on various colorimetric analyses, and can be used for POC assays of many other substances. It should be noted that although the volume of the reaction chambers in the urinary protein quantification chip was on the order of several mm3 because of on-chip dilution, considering the dimension of all the channels printed here were on the order of submillimeters and the majority of the literature reported such submillimeter 3D printed devices as microfluidic devices,9,13,14 in the following sections, we will describe our chip as microfluidic rather than millifluidic devices.

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METHODS AND MATERIALS

3D Printing of Microfluidic Chips. All microfluidic chips and components were designed in AutoCAD and printed by Miicraft 3D printer with clear resin (BV-003). The 3D printing mechanism and procedures are described elsewhere.14 Briefly, it is based on microstereolithography (SL), which contains a digital micromirror device (DMD) to project UV patterns onto the resin (Figure S-1a). After exposure for 7 s, a single layer of resin was cured. Then the motorized stage moved the substrate upward for 100 μm to print another layer of resin. By repeating these steps, microchips were printed using a layer-by-layer process (Figure S-1b). All freshly printed chips and components were soaked in industrial grade ethanol for 5 min to remove uncured resin (rinsing solvent can be ethanol15 or isopropanol14), air-dried, and UV postcured for 10 min in the postcure chamber of the 3D printer (Figure S-1c). According to the manufacturer, the clear resin (BV-003) is acrylate based with a composition of modified acrylate oligomer and monomer, an epoxy monomer, a photoinitiator, and additives. Sealing of Movable Parts. Since the resolution of the 3D printer is about 50 μm and the material is inelastic, a sealing compound must be used to prevent leakage. Vaseline was applied on all of the contacting surfaces between movable parts and the chip to create this seal. Although the sealing Vaseline may contact with the samples or reagents, it is believed that Vaseline does not cause interference. This is because Vaseline is a mixture of long hydrocarbon chain that has no functional group which is inert in terms of chemical activity. Choice of Total Protein Assay. The total protein assay reagent was purchased from Bio-Rad. It consists of Coomasie Brilliant blue G250 dye, for which the absorption peak shifts from 465 to 595 nm when it reacts with proteins. By determining the absorbance, the protein concentration can be quantified with a calibration curve. The

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RESULTS AND DISCUSSION 3D Printed Movable Chip Components. Fluid manipulation on microfluidic chips is mostly achieved by components such as pumps and valves, and has laid the foundation for current microfluidic technology. In this part, we demonstrate one type of pump and three types of valves, which aim to provide specific tools to manipulate fluid without the needs of

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ACS Sensors off-chip equipment. It is important to note that all of the components described here are designed to be extremely simple and convenient for an end-user and are meant to be used by the general public without prior knowledge about microfluidics. As a trade-off, some other advantages of microfluidics may be compromised (which are discussed in the corresponding sections below); however, the overall performance of these components can satisfy the needs of point-of-care devices. Torque-Actuated Pump. Delivering solution to the right location is the primary step of microfluidic operations. Conventionally, for lab-on-a-chip purposes, solutions are commonly pumped through syringe pumps, gas pressure lines, or electroosmotic force because of their convenience in the laboratory setting and ease of controlling flow parameters such as flow rate with high programmability. However, in nonresearch settings (e.g., bedside), such equipment is not readily available and the cost is too high for the general public, which limits the commercialization of microfluidics especially in POC markets. Hence, many pumping mechanisms and components are developed to avoid bulky equipment.23,31−35 Among them, some are relatively simple (e.g., capillary-based pumping23), while others are more complicated but offer more functions (e.g., hand-held gas pressure generator35). The optimal pumping approach should be the simplest method that offers the essential functions. In POC diagnostic setting, the most critical function of a pump is the capability of delivering the sample to the right location that contains the assay reagent. Here we report a simple design for a pump, which is user-friendly and can be easily made by 3D printing. The pump consisted of two parts: (1) the screw and (2) the main chip piece (Figure 1a). The top part of the screw was designed for easy handling (by bare hands or screwdriver), while its middle part was threaded, and the lower part was a cylinder, which acts as a piston. For the main chip piece, it contained a threaded part, a solution reservoir, and a channel (see Figure S-4 for the close-up details of both the male and female threads). Here, the size of the piston and the solution reservoir determined the maximum volume that the pump can deliver. To demonstrate its operation, the reservoir was filled with a blue aqueous dye solution, and then the sealing Vaseline was applied on the screw and it was assembled into the chip. It can be rotated in a clockwise (tightening the screw) direction to deliver liquid from the reservoir into the channel (Figure 1b and Video S-1); interestingly, by rotating the screw in the opposite direction (i.e., anticlockwise), the solution in the microchannel was withdrawn to the reservoir. Because of manual operation, this pump sacrifices accurate control of the flow rate but its simplicity and low manufacturing cost make it particularly suitable in POC applications that just require the delivery of solution to a particular location. However, if the control of flow rate is highly desired in a particular POC application, simple equipment such as motor could be used to rotate the pumping unit. The flow rate of the fluid can thus be controlled by the rotating speed of the motor. Torque-Actuated Valve. By reducing the size of the piston, the torque design can function as a valve as shown in Figure 1c. In the open state, the piston stayed out of the channel, while, by rotating the valve in clockwise, the piston blocked the vertical channel entirely to close the valve (Figure 1d). The thin layer of sealing Vaseline that was applied on the surface of the piston can help to avoid liquid leakage. For characterization, the entire chip was loaded with water; then the

Figure 1. Torque-actuated chip components. (a) Schematic representation of the pumping unit. (b) Photograph of 3D printed pumping unit. It is loaded with blue dye solution. (c,d) Schematic illustration of the torque-actuated valve: valve opened (c), valve closed (d). (e) Fluorescent image of the 3D printed valve unit. It was observed from the bottom and the central dashed white circle depicts the position of the valve. (f) Fluorescent image of (e) after 4800 s.

valve was closed, followed by continuously injecting the fluorescein sodium solution into the chip at 1 μL/min through the inlet by a syringe pump. Figure 1e shows that after 80 min, no fluorescein diffused across the valve, proving that the valve functioned properly. Compared with existing microfluidic valves such as Quake’s valve,3 our design does not require external power or gas pressure sources to operate or maintain closed. Although this approach is not suitable for a large number of valves (due to its footprint size) or perform complicated valving, it fits well in portable and disposable microfluidic applications that require simple on/off microfluidic operations. Rotary Valve. Inspired by the valve commonly used in high-pressure liquid chromatography and gas chromatography, we fabricated a rotary valve (Figure 2a) that can be used in 3D printed chips, particularly suitable for transferring liquids with accurate volumes. The valve consisted of a top part for handling and a bottom part containing fluid channels (coated with Vaseline). In this demonstration, one channel was loaded with the fluorescein sodium solution while the other channel was loaded with water (Figure 2b,c). After loading, the valves could be rotated so that the two solutions made contact with each other and started to diffuse (Figure 2e,f). Here, since the channels had a width of 500 μm, the alignment was done by observing with naked eyes. However, to simplify the alignment process in practical situations, blocking posts can be included on both the chip and the rotary valve. As a result, once the posts block each other from further rotation, the users can sense that the alignment is completed. 229


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printing area, manufacturing time per chip could be further reduced by printing multiple chips simultaneously. Compared with the rotary valve, the pushing valve is easier to operate and its single-use design only requires the users to push the valves all the way to the end. In this manner, the error caused by manual operations is reduced. Hence, it was chosen to be the sample delivery method used in the following integrated chip for POC diagnosis. Integrated Chip for POC Diagnosis. We chose to integrate the torque-actuated pump and the pushing valve developed above for a disposable POC diagnostic device that can be used for general colorimetric chemical assays. Since urine can be collected noninvasively and it carries rich information about the human body, especially for the kidney,24 measuring total protein content in urine was selected as the proof-of-concept model for this device. Chip Design. Generally, the linear range of colorimetric assay reactants is limited and does not cover the entire concentration range of interest of the target, so sample dilution is often required. This is also true for the assay reactant used in our experiment. In order to determine the concentration of urinary protein within the physiologically relevant linear range, three sample dilutions were made with a dilution factor (D.F.) of 1.4, 10, and 50. This was done on chip by controlling the volume ratio between the reaction chambers (loaded with assay reactants) and channels in pushing valves (loaded with sample). The chip design is shown in Figure 4. It consisted of four testing zones and one torque-actuated pump. Among the testing zones, the left (D.F. = 1.4) corresponds to quantifying urinary protein within the range of 0.025−0.20 mg/mL. The middle two zones (D.F. = 10, corresponding to 0.20−1.5 mg/ mL) are identical to each other, giving duplicate assay results. The right zone (D.F. = 50) is designed to quantify highly concentrated protein (1.0−6.0 mg/mL) in urine. In this assay format, the total volume of assay reagent used for the four testing zones is 15 μL which is much less than the conventional microtiter plate based platform (about 800 μL of assay reagent for four tests). Beside sample dilution, controlling the optical path is also important for colorimetric assay. A longer optical path can lead to higher sensitivity. Here, the optical path of the assay was controlled by the height of the reaction chamber and it was fixed to 1 mm across all testing zones. With this length, the assay can be performed at acceptable sensitivity without dedicated detector while the chip consumes less material and can be printed within a shorter period. Manufacturing Process. After the chip and its components were 3D printed, they were assembled as shown in Figure 4a. First, a thin layer of Vaseline was applied on the sidewall of the pushing valve and the torque-actuated pump. Then the valve was inserted into the chip, assay dye solution was loaded into the reaction chamber, and pistons were assembled to confine the volume and block the outlet of the reaction chamber. At this stage, the chip is preloaded with reagents and ready for delivery to the end-users with proper packaging. End-User Operation. In this demonstration, artificial urine spiked with BSA was used as sample. The “end-user operation” is shown in Figure 4b and Video S-2. First, a drop of sample was loaded in the sample reservoir by a dropper. Then the sample solution was pumped into the chip by pushing and rotating the torque-actuated pump. Next, the valves were pushed into the chip so that the sample contacted with the assay reactants. After 25 min of reaction incubation, the assay

Figure 2. Rotary valve. (a) Schematic representation of the valve design. The valve is embedded with channels. (b) Photograph of the 3D printed chip. Fluorescein sodium solution was loaded to the top channel while the bottom channel was loaded with water. (c−f) Fluorescent image of the 3D printed chip. White dashed line depicts the location of the water filled channel; initial state (c); rotated 30° (d); rotated 115° and diffusion started (e); diffused for 20 min (f).

Pushing Valve. Furthermore, we developed another type of valve that can be operated in a simpler manner than the rotary valve. Figure 3a shows the design of the valve (the chip contained 6 identical valves), assembling procedures, and operation. After inserting the valves into the chip, the top channels were connected to form a continuous fluidic path that enabled loading of fluorescein sodium solution into the top channel. Next, water was added to the reaction chamber through the inlet located on the pushing valve. In order to confine the volume of the chamber and block the outlet, rectangular pistons were inserted to the chip after the loading of water (the fluorescent image of the chip at this stage is shown in Figure 3b). Finally, the valves were pushed toward the chip by fingers so that the fluorescein sodium solution loaded in the valves moved downward to make contact with the water in the reaction chamber. The fluorescein solution was mixed with water by diffusion (Figure 3c). The concept of manually moving chip components to achieve controlled mixing is similar to the SlipChip technology.36−39 Although many dedicated applications of SlipChip have been demonstrated, the major hindrance of its widespread adoption is that the device fabrication relies on standard photolithographic and glass etching process, which is time-consuming, labor intensive, and hazardous. In contrast, 3D printing is quicker and more convenient. One chip with all components can be made in less than 20 min, including postcuring time. It should also be noted that with a manufacturing grade 3D printer that has a larger 230


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Figure 3. Pushing valves. (a) Schematic representation of the assembling and operation of the pushing valves. The valves and pistons are colored in red and yellow, respectively. (b) Fluorescent image of the assembled chip loaded with fluorescein sodium solution and water, white dashed lines indicate the valves, piston, and water filled reaction chamber. (c) Fluorescent image of the chip after pushing in the valves for 15 min. The fluorescein sodium molecules diffused to the reaction chamber.

dye solution was fully developed (Figure S-5) and inserted into the chip holder of the smartphone-based platform to quantify the urinary protein. In a practical situation, a demonstration video can be played on the users’ smartphone to assist the operating process. In this design, the goal of the pump is to load the sample into the channel of the pushing valves. As a result, instead of using the torque-actuated pump, it is also possible to deliver the sample with a hand-held plastic syringe with a tube connecting to the chip. However, the volume of the smallest hand-held plastic syringe commercially available is 1 mL, which is difficult to manipulate and deliver just about 15 μL of sample into the chip. With glass syringes that are designed to handle such a small amount of fluid, the cost is very high. In the case of urine, because it is abundant, it is acceptable to apply the plastic syringe approach. The user can load a lot more than the required volume of urine (e.g., 500 μL) into the plastic syringe, followed by injection to fill the channels inside the chip. However, for more precious samples, such as blood, this approach is impractical. Since the objective of this work is to develop a general colorimetric analytical platform, we would like to demonstrate with the torque-actuated pump which can handle both the abundant and precious samples. To ensure that the chip functions well and reduce the chance of false negatives, the user needs to pay attention to the testing

zone corresponding to low protein concentration (the leftmost zone). Once the valve is pushed in, the original red color of the assay reactant in the leftmost reaction chamber fades out immediately because of dilution (Video S-2). Also, after reaction incubation, the user can see pale blue in the reaction chamber because of the proteins naturally present in normal urine.24 The appearance of the colors indicates that the chip functions well and all the other valves are loaded with the sample properly. Urinary Protein Quantification. With the aid of smartphone-based detection platform, three calibration curves corresponding to three concentration ranges were plotted (Figure 5). The urinary protein content should be less than 0.1 mg/mL in a healthy adult.40 Here, we can quantify the concentration of urinary protein with a range from 0.025 mg/ mL to 6.0 mg/mL linearly. The limit of detection (LOD) was calculated to be 8.5 μg/mL, which is well below the normal value (0.1 mg/mL). To distinguish different renal diseases such as nephrotic syndrome ([protein] > 2.3 mg/mL), subnephrotic range proteinuria (0.66 mg/mL < [protein] < 2.3 mg/mL), clinical albuminuria (0.27 mg/mL < [protein] < 0.66 mg/mL) and microalbuminuria (0.1 mg/mL < [protein] < 0.27 mg/ mL),23,24 an assay with dynamic range of 0.1−2.3 mg/mL of urinary protein is required. The linear range (0.025−6.0 mg/ mL) of our chip is sufficient to distinguish these diseases 231


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Figure 4. Schematic representation of the integrated chip for urinary protein quantification. (a) Manufacturing process. (b) Steps of end-user operation. The stock concentrate of the assay reactant was diluted to 1/2.5 (for low concentrated protein detection chamber, red) and 1/5.5 (for medium and high concentrated protein detection chamber, pale green), respectively, prior to being loaded in the reaction chambers. (c) Photograph of the sample loaded chip. (d) The valves were pushed into the chip in order to mix the sample with the assay reactant by diffusion. (e) The smartphone-based detection platform for quantifying total protein amount in the sample. The light shield and its lid are used to block surrounding light. (Inset) The image of the core setup, which was placed inside the light shield.

expensive part, cost about US$6) and is suitable for point-ofcare devices.

although reports revealed that a few severe patients could excrete more than 6 mg/mL urinary protein.41,42 Compared with recent paper-based microfluidic reports that have quantified urinary protein, our method offers the following advantages: (1) much broader linear range because of on-chip dilution;23,43 (2) longer optical path (1 mm versus 10 μm21), which means better sensitivity; (3) better homogeneity of color distribution;22 and (4) better performance on assay reagents that would interact with cellulose presented in paper (e.g., the assay reactants used in this work). Because most test strips on the market are designed to give qualitative (e.g., pregnancy test strips) or semiquantitative (e.g., pH indicating and urine test strips) results, the developed chip can perform a fully quantitative analysis, which is more informative. We envision that such a fully quantitative format can eliminate the need for retesting in the central laboratory, which is commonly required when using urine test strips. Finally, it should be noted that although the model smartphone used was iPhone 5S, other brands and even entry-level smartphones with cameras can also be used to acquire images. Also, the smartphone accessory is required to aid in quantifying the urinary protein. However, compared with the conventional equipment to operate microfluidic chip such as syringe pump and gas pressure sources, our accessory is much lighter and cheaper (for the lens, which is the most

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CONCLUSIONS In this work, we developed the 3D printed torque-actuated pump and valve, rotary valve, and pushing valve for the first time. All of these components can be manually operated and do not require external bulky equipment to function. The integrated microchip is a general platform for colorimetric quantitative assay. Assays for different substances can be performed with their corresponding assay reagents; if needed, the volume ratio of assay and sample solution, and optical path of the reaction chamber, can also be modified conveniently by 3D printing. Together with the smartphone-based detection system, the developed 3D printed microfluidic chips can enjoy the benefits of reduced reagent consumption, on-site quantitative detection, and easy operation protocol with low manufacturing cost (the chip used in urinary protein quantification cost about US$0.22). Depending on the assay reagents selected, sensitive and selective analyses can be performed. Although the developed device is still more complicated and expensive to operate than urine test strips and some of the paper-based microfluidic devices, in return, the developed chip can provide richer quantitative information. This additional quantitative information is useful in diagnosis and potentially eliminate the needs of repeated quantification in 232


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developed format is still far cheaper than conventional central facilities based analysis. Currently, the 3D printed microchip is still in the early development stage. The 3D printing industry is growing rapidly and we can foresee that functionalities and complexities of 3D printed chips will expand along with the development of the 3D printing technique, such as improvement in resolution and multimaterial printing which can integrate electrode and elastic material on a chip. These advancements can potentially increase the throughput and enrich the functions of 3D printed chips, which are essential in developing the lab-on-a-chip device that the general public can benefit from in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00100. Figures of the 3D printing process; figures of the smartphone quantification accessories; figures of image processing; figures of the details of threads in the torqueactuated pump; figure of the incubated urinary protein quantification chip (PDF) Video of operation of the torque-actuated pump (MOV) Video of the “end-user-operation” of the urinary protein quantification chip (MOV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel:+852-23587246. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Hong Kong Research Grant Council (GRF#604712). REFERENCES

(1) Why 3D Printing Could Be A Manufacturing And Logistics Game Changer; http://www.manufacturing.net/blogs/2013/10/why3d-printing-could-be-a-manufacturing-and-logistics-game-changer (accessed Feb 18, 2015). (2) Au, A. K.; Lee, W.; Folch, A. Mail-Order Microfluidics: Evaluation of Stereolithography for the Production of Microfluidic Devices. Lab Chip 2014, 14, 1294−1301. (3) Unger, M. A.; Chou, H.-P.; Thorsen, T.; Scherer, A.; Quake, S. R. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science (Washington, DC, U. S.) 2000, 288, 113−116. (4) Weibel, D. B.; Kruithof, M.; Potenta, S.; Sia, S. K.; Lee, A.; Whitesides, G. M. Torque-Actuated Valves for Microfluidics. Anal. Chem. 2005, 77, 4726−4733. (5) Zheng, Y.; Dai, W.; Wu, H. A Screw-Actuated Pneumatic Valve for Portable, Disposable Microfluidics. Lab Chip 2009, 9, 469−472. (6) Ma, H. K.; Hou, B. R.; Wu, H. Y.; Lin, C. Y.; Gao, J. J.; Kou, M. C. Development and Application of a Diaphragm Micro-Pump with Piezoelectric Device. Microsyst. Technol. 2008, 14, 1001−1007. (7) Anderson, K. B.; Lockwood, S. Y.; Martin, R. S.; Spence, D. M. A 3D Printed Fluidic Device That Enables Integrated Features. Anal. Chem. 2013, 85, 5622−5626. (8) Chen, C.; Wang, Y.; Lockwood, S. Y.; Spence, D. M. 3D-Printed Fluidic Devices Enable Quantitative Evaluation of Blood Components in Modified Storage Solutions for Use in Transfusion Medicine. Analyst 2014, 139, 3219−3226.

Figure 5. Quantification results of each concentration range of urinary protein (n = 6, error bar = ±SD). (a) 0.025−0.20 mg/mL; (b) 0.20− 1.5 mg/mL; (c) 1.0−6.0 mg/mL. (Inset) The gray scale image of the reaction chamber mixed with sample with different concentrations of protein. The scale bar is 1 mm.

a central laboratory. We believe that paper-based device will continue to dominate entry-level analysis because of its low cost. However, when the application requires higher analytical performance such as better linearity, wider dynamic range, and higher sensitivity, the developed device which is based on the conventional absorbance-based analysis will become more competitive and eventually offset the disadvantage of being higher in cost. Finally, it should be noted that the analysis in the 233


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