Siphon-Controlled Automation on a Lab-on-a-Disc Using Event-Triggered Dissolvable Film Valves

Within microfluidic technologies, the centrifugal microfluidic “Lab-on-a-Disc” (LoaD) platform offers great potential for use at the PoC and in low-resource settings due to its robustness and the ability to port and miniaturize ‘wet bench’ laboratory protocols. We present the combination of ‘event-triggered dissolvable film valves’ with a centrifugo-pneumatic siphon structure to enable control and timing, through changes in disc spin-speed, of the release and incubations of eight samples/reagents/wash buffers. Based on these microfluidic techniques, we integrated and automated a chemiluminescent immunoassay for detection of the CVD risk factor marker C-reactive protein displaying a limit of detection (LOD) of 44.87 ng mL−1 and limit of quantitation (LoQ) of 135.87 ng mL−1.


Introduction
Cardiovascular disease (CVD) encompasses a large array of disorders. The most common is arteriosclerosis which is the buildup of plaque on the arterial wall, restricting the flow of oxygen to different tissues in the body. CVD is among the primary causes of death worldwide with an estimated 15.5 million incidences in 2012 and is becoming more prevalent with increasing standards of living across the global population [1]. Heart failure has emerged as a particularly prevalent diagnosis in sub-Saharan Africa [2], but other often under-researched conditions, which are relatively limited to tropical regions, such as endomyocardial fibrosis (EMF) and rheumatic heart disease, are becoming more prevalent [1]. Among mitigation strategies proposed to address these issues are the implementation of field-friendly Point-of-Care (PoC) technologies to assist diagnosis [1,3]. To address this need, lab-on-a-chip devices can provide bioanalytical tests on patient samples [4][5][6][7].
A wide range of biomarkers have been associated with CVD, including creatine kinase (CK), creatine kinase-MB (CKMB), myoglobin, and C-reactive protein (CRP) as well as cardiac troponin I and T. Typically, after a cardiovascular event that restricts blood flow to the tissue, these molecules are released into the blood from dying cells. However, as these molecules can also be present in other tissues, there can be considerable variability in predictive power of these markers for CVD [8]. Of identified biomarkers, CRP has been modulating the spin rate, with a geometry-dependent threshold frequency for opening. Active valves rely on interaction with an instrument-based unit (often integrated into the centrifugal spin-stand). A third, less common, valving technology is based on the timed disintegration of (water) dissolvable films (DFs) and/or liquid movement [38,39]. A comparison of different valving technologies, particularly as related to passive flow-control and to the dissolvable film valves used in this study, is provided in Table 1.
at a constant disc speed.
Dissolvable Film (Event-triggered with instrumentation) incorporates the event-triggered architecture except the actuation of valves is through external actions such as piercing a tape or melting a wax film.
Permits complex multi-step assays (60+ steps). Suitable for high disc spin-speeds. Feedback control possible.
Requires multilayer architecture. Requires embedded DF valves. No long incubations/washes. Single use valves. Requires support instrumentation, [29,61] Dissolvable Film (Event-triggered with Siphon Control) are described in Figure 1 Permits complex multi-step assays. Suitable for high disc spin-speeds. Timing of valve opening/incubations using only rotational control.
Requires multilayer architecture. Requires embedded DF valves. Single use valves (except siphon).
- Figure 1. Event-triggered sequential release of centrifugo-pneumatic siphon valves (CPSVs). (a,b) Loading of sample (green), at a high spin rate, into the incubation chamber. Alternating the spin rate can be used to induce mixing in the incubation chamber. (c) At a low spin rate, the centrifugo-pneumatic siphon valve is primed and (d) then emptied at a medium spin rate. (e) The control film (CF) of the following valve is wetted to vent the pneumatic channel release to the next reagent (blue). This process can be repeated to control the timed release and incubation of further reagents.
The focus of this work was an advancement of the event-triggered dissolvable films valves (Table 1), whereby their performance is enhanced through coupling them with a centrifugo-pneumatic siphon valve. Event-triggered valves function akin to single-use electrical relays. The arrival of liquid at a first DF, referred to as the control film (CF), triggers the release of a liquid at a second DF at a distal location, referred as the load film (LF). Properly configured, this coordinates the release of liquid bioreagents in a well-defined sequence. This valving technology offers independence from the spin rate (as valve actuation is governed by DF dissolve time and liquid movement) and the limiting factor on the number of LUOs/valves in sequence is only the available space on the disc.
A critical limitation of event-triggered valving is that timing of their actuation depends on the dissolution time of the DFs and intervals for liquid transfer about the disc. While their dissolution time can be tailored by their formulation [62][63][64] and even used for reagent storage [65,66], resorting to commercially available (single-grade) DFs in microfluidic devices is beneficial toward mass manufacture [67]. To control timing of event-triggered valves, researchers have focused on alternative mechanisms such as hy- Figure 1. Event-triggered sequential release of centrifugo-pneumatic siphon valves (CPSVs). (a,b) Loading of sample (green), at a high spin rate, into the incubation chamber. Alternating the spin rate can be used to induce mixing in the incubation chamber. (c) At a low spin rate, the centrifugo-pneumatic siphon valve is primed and (d) then emptied at a medium spin rate. (e) The control film (CF) of the following valve is wetted to vent the pneumatic channel release to the next reagent (blue). This process can be repeated to control the timed release and incubation of further reagents. Table 1. Comparison of common lab-on-a-disc valving technologies.

Name and Operation
Advantages Disadvantages Refs Capillary Valves are actuated by increasing the disc spin-rate. They function based on the balance of body forces (governed by relative centrifugal force) and interfacial tension holding the liquid in place.
Simple operation and ease of manufacture.
Cannot operate at high disc speeds. Highly dependent on manufacturing fidelity. Low number of assay steps. [40,41] Capillary-action primed siphon valves are low-pass valves. Thy are triggered by reducing the disc spin-rate which allows capillary priming of a siphon. They can be combined in series (with capillary valves) to enable actuation by sequentially increasing and decreasing disc spin speed.
Simple operation and ease of manufacture. Can enable sample incubation.
Highly dependent on manufacturing fidelity. Low number of assay steps. Can use significant disc real-estate. [32,[42][43][44][45] Centrifugo-pneumatic siphon valves (CPSV) function in a manner similar to siphon valves except the release of compressed air (trapped during loading of a reservoir) primes the siphon rather than the capillary force.
Simple operation and ease of manufacture. Can enable sample incubation. Reliable and tolerant to low-fidelity manufacture.
Highly reliable. No external instrumentation required-rotational control only.
Can require a powerful motor to generate necessary acceleration (Euler Force). Can use significant disc real-estate.
Requires multilayer architecture. Requires embedded DF valves. Single use valves (except siphon).

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The focus of this work was an advancement of the event-triggered dissolvable films valves (Table 1), whereby their performance is enhanced through coupling them with a centrifugo-pneumatic siphon valve. Event-triggered valves function akin to single-use electrical relays. The arrival of liquid at a first DF, referred to as the control film (CF), triggers the release of a liquid at a second DF at a distal location, referred as the load film (LF). Properly configured, this coordinates the release of liquid bioreagents in a welldefined sequence. This valving technology offers independence from the spin rate (as valve actuation is governed by DF dissolve time and liquid movement) and the limiting factor on the number of LUOs/valves in sequence is only the available space on the disc.
A critical limitation of event-triggered valving is that timing of their actuation depends on the dissolution time of the DFs and intervals for liquid transfer about the disc. While their dissolution time can be tailored by their formulation [62][63][64] and even used for reagent storage [65,66], resorting to commercially available (single-grade) DFs in microfluidic devices is beneficial toward mass manufacture [67]. To control timing of event-triggered valves, researchers have focused on alternative mechanisms such as hybrid event-triggered valves that are opened using supporting instrumentation [61]. Another approach, and the focus of this article, is to place a reusable and rotationally controlled valve (i.e., a siphon valve [45] or centrifugo-pneumatic siphon valve [50,51]) in the flow path for on-disc flow control. This strategy allows the disc architecture to govern the sequence of sample/reagent release while changes in spin rate control the timing of reagent release. This platform establishes arbitrarily defined (i.e., programmable via spindle motor) incubation periods that are often required in typical assay protocol.
Chemiluminescence (and chemiluminescent immunoassays) have been previously demonstrated within lab-on-a-chip devices [68][69][70][71][72][73] and, specifically, on LoaD platforms [9,[74][75][76][77]. In this work, we demonstrated detection of CRP, from buffer, using a chemiluminescent immunoassay in the clinically relevant range. Note that in many assays, such as the one described here, samples are diluted in dilute inhibitory proteins/molecules. In the case of CRP, concentrations in blood are typically in the µg mL −1 range but, due to the dilution associated with this assay, the detection levels measured are in the range of ng mL −1 .
The fluidic architecture presented in this manuscript, which combined a centrifugopneumatic siphon valve with event-triggered valves, unprecedentedly demonstrated full fluidic control, with arbitrarily timed incubations, of eight different reagents. Using off-disc measurements, our system showed a linear relationship (R 2 = 0.91) between concentration and luminescent intensity in a range of measurements (n = 3) made between 0 to 80.5 ng mL −1 The limit of detection was 29 ng mL −1 , which represents patients at the threshold of the high-risk category of cardiovascular disease.

Disc Architecture
In this paper, we demonstrate robust, highly multiplexed rotational flow control of one capillary valve and seven event-triggered DF valves. Incubation intervals are defined by changes in the spin rate of a programmable spindle motor. This was enabled by placing an incubation chamber, gated by a centrifugo-pneumatic siphon valve, between the central reagent storage reservoirs and the liquid waste chamber (Figures 1-3). At high spin rates (in this case 40 Hz), the liquid (i.e., sample, reagents, or wash-buffer) enters the incubation chamber and is retained by the unprimed siphon valve. As typical for CPSV valves, liquid is displaced into a dead-end pneumatic chamber, which ensures that the liquid level remains below the siphon crest. As the incubation chamber retains the liquid, downstream CFs are not yet wetted. The spin rate can be varied in the interval between 20 Hz and 40 Hz, which displaces liquid into the incubation chamber and enhances mixing, without priming the siphon valve. Following incubation, the durations of which are described in Figure 4, the spin rate is reduced to 10 Hz, which expels liquid from the pneumatic chamber and increases the liquid level in the incubation chamber. The siphon valve then primes, and liquid is transferred into the waste chamber.  contains the sample, R3 and R4 are filled with washing buffers, R5 contains the detection antibody, R6 and R7 contain wash buffers, and R8 contains the substrate to catalyze chemiluminescent detection. Note, for data presented in Figure 6, R8 was left empty and, following the final washing step (R7), the disc was stopped. The magnets were removed, and the beads were suspended in buffer and aspirated into a pipette through a vent on the incubation chamber. Off-disc measurement was made via GloMax 96 Microplate Luminometer according to the protocol described in ESI.  contains the sample, R3 and R4 are filled with washing buffers, R5 contains the detection antibody, R6 and R7 contain wash buffers, and R8 contains the substrate to catalyze chemiluminescent detection. Note, for data presented in Figure 6, R8 was left empty and, following the final washing step (R7), the disc was stopped. The magnets were removed, and the beads were suspended in buffer and aspirated into a pipette through a vent on the incubation chamber. Off-disc measurement was made via GloMax 96 Microplate Luminometer according to the protocol described in ESI. and R7 contain wash buffers, and R8 contains the substrate to catalyze chemiluminescent detection. Note, for data presented in Figure 6, R8 was left empty and, following the final washing step (R7), the disc was stopped. The magnets were removed, and the beads were suspended in buffer and aspirated into a pipette through a vent on the incubation chamber. Off-disc measurement was made via GloMax 96 Microplate Luminometer according to the protocol described in ESI.
(20-min sonication at 60 °C), followed by washing in deionized (DI) water twice (20-minute sonication at 60 °C), and air drying in a HEPA-filtered assembly room [50,59]. PSA layers were prepared for assembly under sterile conditions; here, the liners are only removed in a High-efficiency particulate air -filtered (HEPA-filtered) cleanroom. The discs were assembled manually on a custom alignment jig. Between addition of each layer, the discs were rolled at least 12 times (three times and four orientations) using a high-pressure laminator (HL-100, Cheminstruments, Fairfield, OH, USA). The waste chamber is segmented into volumes that are equal or slightly less than the volume of liquid released from the incubation chamber. Each segment of the waste chamber contains a CF, which, when wetted, will open an event-triggered valve. Thus, the transfer of liquid to the waste chamber prompts the release of the next liquid defined in the protocol. This combination of the event-triggered valving with the centrifugopneumatic siphon enables arbitrarily timed reagent delivery and so establishes freely definable incubation intervals.

Disc Manufacture and Assembly
The discs were designed with SolidWorks (Dassault Systèmes, Paris, France) as a 3D structure and then 2D AutoCAD Drawing Exchange Format (AutoCAD DXF) files were extracted from this model (Figure 2). Individual layers were then machined based on these drawings. Poly-(methylmethacrylate) (PMMA) layers were machined from 1.5-mmthick PMMA sheets (Radionics, Ireland) using CO 2 laser ablation (Exilog Zing, Golden, CO, USA). Medical-grade Pressure Sensitive Adhesive (PSA) (ARCare ® 7840, Adhesives Research, Limerick, Ireland) was structured using a knife-cutter (Graphtec CE6000-40, Irvine, CA, USA). The disc was assembled ( Figure 2) from a stack of eight layers: (1) Vent layer of PMMA, containing loading ports/air vents; (2) Microchannel layer of PSA, containing microchannels for reagent and air transport; (3) Reservoir layer of PMMA, containing reagent reservoirs, waste chambers, pneumatic chambers, incubation chamber, and connecting vertical vias; (4) DF cover layer (PSA), which seals DF tabs into the disc; (5) DF support layer (PSA), which provides alignment and mechanical support for DF tabs; (6) Intermediate layer (PMMA) provides mechanical support for DFs; (7) Lower channels (PSA), containing microchannels for reagent and air transport; and (8) Base (PMMA) provides a layer to seal the lower channels. This layer also contains mechanical support for permanent magnets.
In addition to these layers, permanent magnets (S-03-06-N, Supermagnete, Gottmadingen, Germany) are embedded (mechanical fit) into the base layer to provide a point of agglomeration for the paramagnetic beads. Additionally, a loading hole for reagent removal (located in the vent layer) is sealed with adhesive tape. DF tabs were assembled, as described previously [38], from gas-tight, water-dissolvable film (KC-35, Aicello, Aichi, Japan). These tabs take approximately 30-40 s to dissolve in water at room temperature [38] and provide a short time delay between wetting of the DFs in the waste chamber and release of the next reagent.
The layers from which the cartridges were assembled were cleaned prior to assembly. This protocol includes washing the PMMA in dilute Micro-90 ® Concentrated Alkaline Cleaning Solution (International Products Corporation, Burlington, NJ, USA) twice (20-min sonication at 60 • C), followed by washing in deionized (DI) water twice (20-min sonication at 60 • C), and air drying in a HEPA-filtered assembly room [50,59]. PSA layers were prepared for assembly under sterile conditions; here, the liners are only removed in a Highefficiency particulate air -filtered (HEPA-filtered) cleanroom. The discs were assembled manually on a custom alignment jig. Between addition of each layer, the discs were rolled at least 12 times (three times and four orientations) using a high-pressure laminator (HL-100, Cheminstruments, Fairfield, OH, USA).

Centrifugal Test Stand
The discs were characterized on a centrifugal 'test stand' [29]. Here, a computercontrolled spindle motor (FESTO, Esslingen, Germany) was synchronized with an externally triggered CCD camera (Pixelfly, PCO, Kelheim, Germany) and strobe light (BVS II, Polytec, Waldbronn, Germany) so that each image was acquired at the same angular position. Therefore, the disc appeared stationary while rotating at even up to 60 Hz. The motor was controlled by custom software (LabVIEW), which enabled programming a spin profile (Figures 4 and 5).

Results
Images of the operation of this microfluidic architecture, acquired from the test stand, are shown in Figure 5. Video showing the full sequence of valve actuation (albeit, with shortened incubation times and using colored water for better visualization) is provided in ESI Video S1 and ESI Video S2. Videos were post-processed as described in ESI. As can be seen in Figure 5, this microfluidic architecture allowed complete control of incubation times through altering the spin rate to open the valves. The architecture proved to be extremely reliable. Once the manufacturing process was established, 18 of 21 discs functioned as expected under automated control of the spindle motor ( Figure  6b). The on-disc CRP assay was completed (n = 3) at six different concentrations and a limit of detection (LoD) of 44.87 ng mL −1 and limit of quantitation (LoQ) of 135.87 ng mL −1 were established.

Biological Assay Materials
The CRP capture antibody, rabbit polyclonal to C-reactive protein, was purchased from Abcam plc (ab31156, Abcam plc, Cambridge, UK). The detection antibody used was a Horse Radish Peroxidase (HRP), labelled goat polyclonal anti-CRP (PA1-28329, Thermo Fisher, Dublin, Ireland). Note that polyclonal antibodies were chosen over a monoclonal antibody in order to ensure greatest chance of assay success (with minimal knowledge of protein structure) while accepting a decrease in assay sensitivity (compared to monoclonal antibodies). The 2.8-µm superparamagnetic beads (Dynabeads ® M-270 Epoxy, Thermo Fisher, Dublin, Ireland) and reagents required for coupling the capture antibody to the beads were acquired as part of the Dynabeads Antibody Coupling Kit (14311D, Thermo Fisher, Dublin, Ireland). The protein standards used were from the CRP Human Kit for Luminex ® Platform (Catalogue number: LHP0031, Thermo Fisher, Dublin, Ireland). Additional reagents used were taken from the Human Extracellular Protein Buffer Magnetic Reagent Kit Thermo Fisher, Dublin, Ireland, Catalogue number: LHB0001). The substrate used was Pierce™ ECL Western Blotting Substrate (T Thermo Fisher, Dublin, Ireland, catalogue number: 32106).

Benchtop Magnetic Chemiluminescence Assay
The assay was optimized on-bench with a particular focus on reducing the assay time. The total assay time was reduced from 180 minutes (according to the recommended protocols) to 40 min (Figure 6a). sample, is released through a DF valve into the incubation chamber. (c) The 'sample' is transferred to the waste chamber where wetting the next DF releases the first wash buffer.

Results
Images of the operation of this microfluidic architecture, acquired from the test stand, are shown in Figure 5. Video showing the full sequence of valve actuation (albeit, with shortened incubation times and using colored water for better visualization) is provided in ESI Video S1 and ESI Video S2. Videos were post-processed as described in ESI. As can be seen in Figure 5, this microfluidic architecture allowed complete control of incubation times through altering the spin rate to open the valves. The architecture proved to be extremely reliable. Once the manufacturing process was established, 18 of 21 discs functioned as expected under automated control of the spindle motor ( Figure  6b). The on-disc CRP assay was completed (n = 3) at six different concentrations and a limit of detection (LoD) of 44.87 ng mL −1 and limit of quantitation (LoQ) of 135.87 ng mL −1 were established.

Discussion and Conclusions
The efficacy of this disc architecture to implement paramagnetic bead-based chemiluminescent immunoassays for CRP was established. The clinical range of interest for CRP is between 3-80 μg mL −1 for adults, and, in the case of neonatal sepsis, as low as 0.08 μg mL −1 [9]. In this work, we demonstrated the capability to detect as low as 44.87 ng mL −1 in buffer. Typically, in a diagnostic test, plasma or serum is isolated from whole blood and then diluted by a defined ratio. This ensures the assay can deliver results in the clinically relevant range while not delivering a signal at saturation. This dilution also Briefly, all standards were made by diluting CRP stock (CRP Human Kit for Luminex ® Platform (Catalogue number: LHP0031, Thermo Fisher, Dublin, Ireland)) with 1X Incubation buffer (Human Extracellular Protein Buffer Reagent (Catalogue number: LHB0001), Thermo Fisher, Dublin, Ireland) to the appropriate concentration. Then, 20 µL of primary antibody-coated Dynabeads ® (10 mg/mL) were placed in each well of a white, 96-well plate. Using a small neodymium magnet, the Dynabeads were held in place while the liquid was removed and discarded. Each well was then blocked, using a 1% solution of BSA in 0.1 M PBS for 1 h at 4 • C. As before, the contents of the well were discarded while the magnetic microparticles were held in place with a magnet. Then, 100 µL of each CRP standard was added to the corresponding well and incubated at room temperature for a given incubation time (standard incubation 2 h).
Each well and microparticle content were then washed twice with 200 µL of wash solution (Human Extracellular Protein Buffer Reagent (Catalogue number: LHB0001), Thermo Fisher, Dublin, Ireland). Then, 200 µL of detection antibody (PA1-28329, Thermo Fisher, Dublin, Ireland) (1/10,000 dilution in 0.1M PBS) was added to each well and incubated for a given incubation time in the dark (standard incubation 1 h). Each well and microparticle contents were then washed twice with 200 µL of wash solution, as before. Just prior to plate reading, the final wash solution was removed from each well and 100 µL of Pierce™ ECL Western Blotting Substrate (Catalogue number: 32106, Thermo Fisher, Dublin, Ireland) was added to each well. Wells were read using a GloMax 96 Microplate Luminometer. See also ESI.

Lab-on-a-Disc Magnetic Chemiluminescent Assay
Prior to their loading to the disc, the magnetic beads were prepared on-bench with the capture antibody according to the protocols described in Section 2.5.
Prior to being placed on the centrifugal test stand, each disc cartridge had two neodymium magnets placed in precut holes behind the incubation chamber. Next, each reservoir was loaded with 90 µL of a specific reagent, corresponding to a step of generic immunoassay.
On-disc reservoirs were filled according to Table 2. R2 contained the specific CRP standards at differing concentrations (80, 40, 20, 10, 5, or 2.5 ng mL −1 ), depending on the concentration of standard being tested. R8 was left empty during assay testing to enable off-disc measurement. See also ESI.

Automated Lab-on-a-Disc Protocol
A pre-programmed protocol was executed unsupervised on the centrifugal test stand. Mixing cycles refer to the number of times the disc was accelerated between 20 Hz and 40 Hz to induce advective mixing. Note the chemiluminescence substrate was not loaded on-disc.
After completion of the automated spin protocol, the disc was removed from the test stand and the magnets were removed from the underside of the disc. Next, the scotch tape was stripped from over the incubation chamber. Then, 60 µL of 0.1M PBS was pipetted into the incubation chamber and the magnetic beads were suspended in the buffer. The buffer and beads were then pipetted from the disc and transferred to a well plate. A magnet retained the beads while supernatant was extracted. Next, 90 µL of 0.1M PBS was loaded into the microtiter plate for resuspending the beads. Under low-intensity illumination, 100 µL of Pierce™ ECL Western Blotting Substrate reagent was added to each the well on the well plate and chemiluminescent signal was measured on a luminometer (GloMax Microplate Luminometer, Promega, Madison, WI, USA).

Results
Images of the operation of this microfluidic architecture, acquired from the test stand, are shown in Figure 5. Video showing the full sequence of valve actuation (albeit, with shortened incubation times and using colored water for better visualization) is provided in ESI Video S1 and ESI Video S2. Videos were post-processed as described in ESI. As can be seen in Figure 5, this microfluidic architecture allowed complete control of incubation times through altering the spin rate to open the valves. The architecture proved to be extremely reliable. Once the manufacturing process was established, 18 of 21 discs functioned as expected under automated control of the spindle motor (Figure 6b). The on-disc CRP assay was completed (n = 3) at six different concentrations and a limit of detection (LoD) of 44.87 ng mL −1 and limit of quantitation (LoQ) of 135.87 ng mL −1 were established.

Discussion and Conclusions
The efficacy of this disc architecture to implement paramagnetic bead-based chemiluminescent immunoassays for CRP was established. The clinical range of interest for CRP is between 3-80 µg mL −1 for adults, and, in the case of neonatal sepsis, as low as 0.08 µg mL −1 [9]. In this work, we demonstrated the capability to detect as low as 44.87 ng mL −1 in buffer. Typically, in a diagnostic test, plasma or serum is isolated from whole blood and then diluted by a defined ratio. This ensures the assay can deliver results in the clinically relevant range while not delivering a signal at saturation. This dilution also improves the accuracy of the assays by diluting components of blood, which may inhibit the assay performance. Implementing this full protocol on disc, including dilution of sample, will be subject to future work. Indeed, integration of this microfluidics architecture with wireless chemiluminescent sensing [74] offers the potential to provide for full sample-to-answer automation of the assay on a compact laboratory instrument. Figure 6b shows measurements across a range of concentrations with both standard and optimized incubation times. These parameters were then used on disc and reduced the overall assay time from over 2 h to approximately 55 min. These same incubation times were then used for the on-disc protocol. However, while measured on the same laboratory instrument, the signals measured from the beads processed on disc were significantly lower than those processed on bench ( Figure 6).
We identified two potential causes of this reduced signal. In the first, the removal of beads from the disc for measurement can result in loss of material. This loss may be mitigated through integration of chemiluminescence measurement into the test-stand instrument [74]. The second problem concerns the use of magnetic beads which, in this implementation, are pulled to the surface by permanent magnets embedded into the underside of the disc. Ideally, this would create a monolayer of functionalized beads to replace an antibody-coated surface [78,79]. A particular advantage of using magnetic beads is the potential to use the same disc architecture against different antibody targets. However, we believe the use of permanent magnets caused unfavorable agglomeration of magnetic beads and, despite rigorous mixing, leaving significant percentage of beads underexposed to sample and/or to detection antibody. This effect may be mitigated through use of an electro-magnet rather than permanent magnets. This would permit the beads to mix freely with the sample/reagents during the incubation steps. Alternatively, the PMMA surfaces inside the disc might be directly functionalized to provide a defined surface for detection [80].
The results presented above clearly demonstrate the capability of this microfluidic architecture to enable biological protocols that require extended incubation periods (i.e., of the order of 20 min and total assay times of more than an hour). This capability allowed us to decouple the timing of release of event-triggered valves from the dissolution time of the DFs (via the use of the siphon structure), but still allow the sequence of valve actuation to be determined by the disc architecture. Thus, this platform has the potential to be applied across a wider range of different biomedical diagnostic applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/2079-6 374/11/3/73/s1. ESI.pdf contains additional information pertaining to the assay optimization and protocols used. Video S1: Optimized Design.mp4 shows the disc design used in these experiments. In this video, colored water was used for visualization, incubations were shortened, and the video was accelerated 8x to assist with viewing. Video S2: Dual Pneumatic.mp4 shows an earlier design used in preliminary testing. Here the centrifugo-pneumatic valve was split into two chambers to better control sample fluid height in the incubation chamber. Video play at 8x normal speed for ease of viewing.