VectorDisk: A Microfluidic Platform Integrating Diagnostic Markers for Evidence-Based Mosquito Control

Effective mosquito monitoring relies on the accurate identification and characterization of the target population. Since this process requires specialist knowledge and equipment that is not widely available, automated field-deployable systems are highly desirable. We present a centrifugal microfluidic cartridge, the VectorDisk, which integrates TaqMan PCR assays in two feasibility studies, aiming to assess multiplexing capability, specificity, and reproducibility in detecting disk-integrated vector-related assays. In the first study, pools of 10 mosquitoes were used as samples. We tested 18 disks with 27 DNA and RNA assays each, using a combination of multiple microfluidic chambers and detection wavelengths (geometric and color multiplexing) to identify mosquito and malaria parasite species as well as insecticide resistance mechanisms. In the second study, purified nucleic acids served as samples to test arboviral and malaria infective mosquito assays. Nine disks were tested with 14 assays each. No false positive results were detected on any of the disks. The coefficient of variation in reproducibility tests was <10%. The modular nature of the platform, the easy adaptation of the primer/probe panels, the cold chain independence, the rapid (2–3 h) analysis, and the assay multiplexing capacity are key features, rendering the VectorDisk a potential candidate for automated vector analysis.

acid purification; and (iii) easy adaptation from Anophelesto Aedes-species-specific assay detection. We assessed the feasibility and reproducibility of the assay integration onto the disk in two small-scale studies: the Vector Study, starting from laboratory-reared mosquitoes, to test the malaria assays; and the Nucleic Acid (NA) Study, starting from purified nucleic acids, to test the arbovirus assays [28]. This proof-of-principle work indicates the potential of the platform to be used for fully automated analysis in the framework of integrated vector control.

Mosquito Assays
In order to demonstrate the capability of the VectorDisk platform to simultaneously collect different molecular information for species identification and insecticide resistance, the assays used in this work were designed to detect: (i) DNA-based mosquito species of the Anopheles gambiae species complex; (ii) if present, the DNA of the malaria parasite, thereby differentiating between P. falciparum (the most lethal for humans) and P. ovale/vivax/malariae [18], and the infective stage through the RNA detection of sporozoite-specific P. falciparum transcripts [29]; (iii) knockdown resistance (kdr) point mutations conferring resistance in the target site of the para voltage-gated sodium channel responsible for the insensitivity to dichlorodiphenyltrichloroethane (DDT) and pyrethroids [30][31][32], as well as target site resistance to organophosphates and carbamates due to insensitive acetylcholinesterase (iAChe) [33]; (iv) overexpression of detoxification loci at the mRNA level [34], which can pinpoint associations with insecticide resistance, such as the cytochrome P450 monooxygenases CYP6P3, CYP6M2, CYP6P4, and CYP6Z1, and the glutathione-S transferase GSTE2 in An. gambiae; (v) the viral RNA, which may be present in arbovirus vectors (i.e., West Nile virus [35] and Zika virus [36]).

Vector Study: Manual Preparation of Mosquito Samples (Lysis and Homogenization)
The insectary at the Swiss Tropical and Public Health Institute provided specimens of the Kisumu colony that originates from an MRA-762 egg batch provided by BEI Resources. For this Vector Study, 50 mosquitoes (Kisumu, 25 male (m) + 25 female (f)) were used, and the following preparatory steps were performed prior to the automated VectorDisk protocol. The mosquitoes were split into five pools of ten mosquitos each (5 males + 5 females). It was shown by Mavridis et al. [31] that the use of pools of ten mosquitoes saves time, reduces costs, and allows for more efficient resistance allele screening than in the case of individual mosquito testing. The mosquitoes were preserved in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA), and thawed on a lint-free towel. They were then washed with ultra-pure H 2 O to remove the RNAlater. Subsequently, they were ground manually in a 150 µL lysis buffer (RLT Buffer, QIAGEN, Hilden, Germany) with a pestle (on ice). The homogenate was incubated for 10 min at room temperature. After centrifugation for 2 min at 16,000 rcf, the supernatant was collected and stored at −20 • C in aliquots. Prior to any next experimental step, the lysate was diluted 1:200 with ultra-pure H 2 O. In order to acquire results that would allow some reproducibility assessment, one lysate from each pool was diluted and aliquoted for testing three to four times. Thus, we could perform inter-pool as well as intra-pool reproducibility assessments. A summary is shown in Figure 1. A total of 18 disks were used in the Vector Study.

Nucleic Acid Study
The study includes the following purified genomic RNA that was obtained through BEI Resources, NIAID, and NIH: genomic RNA from West Nile virus (WNV), Bird 114, NR-9573, genomic RNA from Zika virus (ZIKV), FLR-Zika virus, NR-50241. The RNA concentration was <1.0 ng/µL. For testing the Plasmodium and the infected stage assays (sample coded as 'PF-INF'), we included in the NA Study a mixture of DNA and RNA extracted from laboratory-reared P. falciparum infected mosquitoes as previously described [29] and their concentration was 120 ng/µL. In order to acquire results that would allow some reproducibility assessment, each sample was tested three times in the VectorDisk.

VectorDisk Fabrication
The microfluidic design was created using SolidWorks2017 (Dassault Systèmes SolidWorks Corp., Waltham, MA, USA). Production of the VectorDisk cartridge was done by Hahn-Schickard Lab-on-a-Chip Foundry [37] using rapid tooling and a scalable and low cost micro-thermoforming process for replication of prototypes [38,39] made of Cyclic Olefin Copolymer foil (COC 6013/8007, 200 µm thick, TOPAS Advanced Polymers GmbH, Frankfurt, Germany). For this, an in-house milled (EVO, Kern Microtechnik GmbH, Eschenlohe, Germany) metal master was used on a batch production thermoforming machine (Rohrer AG, Möhlin, Switzerland). No further surface treatment was performed after thermoforming. The primers/probes, together with trehalose as stabilizer (final concentration 50 mM), were pipetted in the reaction chambers, and the cartridges were then left to dry in an oven at 50 • C for 1 h. The combinations of the TaqMan probe assays are shown in Tables 1  and 2. Details on the primer/probe design and validation of the assays, including sensitivity results, are available in previous studies for the malaria-specific [29,31] and arboviral-specific [35,36,40] assays. The primer/probe sequences are given in Supplementary Tables S1 and S2. The structured foil was  sealed with a 200 µm thick COC foil (COC 6013/8007, TOPAS Advanced Polymers GmbH, Germany) using an adaptive thermo-sealing process [41] on a modified hot embossing machine (Wickert GmbH, Landau in der Pfalz, Germany). The sealing foil had an opening at the radius that corresponded to the mixing chamber location, where the lyopellet was inserted manually after the thermal sealing. The lyopellets were developed and supplied specifically for these triplex assays (Fast Track Diagnostics, Luxembourg), and their functionality was tested and validated prior to their use in this work [34]. At the end, a piece of pressure-sensitive adhesive foil 9795R (3M, Maplewood, MN, USA) was used to seal the lyopellet mixing chamber. The cartridges were stored with a desiccant in a sealed aluminium pouch to protect reagents from humidity and light until use. Dengue virus RNA DENV T1-4 --10 Plasmodium species ID DNA P. falciparum P. ovm 1 -11 Plasmodium infective stage RNA --Pf infective stage 1 P. ovm: P. ovale/vivax/malariae, i.e., the primer set detects all three. The symbol '-' means that there were no assay targets expected to be detected in the particular reaction chambers and detection channels.

VectorDisk Workflow
For the Vector Study, upon thawing, the mosquito lysate was diluted 1:200 with ultra-pure H 2 O (RNase/DNase-free). Then, 180 µL of the diluted lysate was inserted into the VectorDisk inlet ( Figure 3A). In the automated process in the LabDisk, 160 µL of diluted lysate was transferred radially inwards (B) to the mixing chamber with the lyopellet (C), using temperature change-rate (TCR) valving [42] and centrifugo-dynamic inward pumping [43]. A dedicated microfluidic protocol was used in chamber (C) for the rigorous and homogeneous rehydration and mixing of the lyopellet, employing temperature change-rate actuated bubble-mixing [44,45]. The mixture was then transferred to the metering fingers (D), and a volume of 10 µL per finger was aliquoted using centrifugo-pneumatic aliquoting [46]. The valving and mixing operations were supported by a dedicated chamber for generating air pressure (E). Then the RT-PCR took place in the reaction chambers (F) where the primers/probes had been dry-stored. The protocol was: 44 • C for 10 min (reverse transcription), 97 • C for 2 min (initial denaturation for the PCR), and 40 cycles of 97 • C for 5 s and 60 • C for 4 s. The structure (F) serves to generate air pressure for valving and mixing operations. The structure (G) serves as a stiffening structure in order to prevent bending of the foil disk.
Processes 2020, 8, x FOR PEER REVIEW 6 of 16 chambers (F) where the primers/probes had been dry-stored. The protocol was: 44 °C for 10 min (reverse transcription), 97 °C for 2 min (initial denaturation for the PCR), and 40 cycles of 97 °C for 5 s and 60 °C for 4 s. The structure (F) serves to generate air pressure for valving and mixing operations. The structure (G) serves as a stiffening structure in order to prevent bending of the foil disk.
For the NA Study, a mixture of 162 µL ultra-pure H2O and 18 µL of purified NA sample was inserted into the inlet. Then, the same fluidic protocol was used as for the Vector Study. At the end, the PCR protocol was started: 50 °C for 15 min (reverse transcription), 95 °C for 3 min (initial denaturation for the PCR), and 40 or 45 cycles of 95 °C for 3 s and 60 °C for 30 s. For both studies, the thermocycling protocol and the microfluidic processing were accomplished by means of a dedicated compact LabDisk Player instrument ( Figure 4, QIAGEN Lake Constance, Stockach, Germany). The detailed microfluidic protocol is described in Supplementary Table S3. Rotational frequency control and air-heating enabled centrifugo-pneumatic [43,46] and thermopneumatic microfluidic unit operations [42,44,47], and allowed for a time of 140-180 min from sample inlet to final result (depending on the Vector or NA Study). PCR raw data acquired with the LabDisk Player were converted and analyzed using the RotorGene Software (QIAGEN, Hilden, Germany). For the NA Study, a mixture of 162 µL ultra-pure H 2 O and 18 µL of purified NA sample was inserted into the inlet. Then, the same fluidic protocol was used as for the Vector Study. At the end, the PCR protocol was started: 50 • C for 15 min (reverse transcription), 95 • C for 3 min (initial denaturation for the PCR), and 40 or 45 cycles of 95 • C for 3 s and 60 • C for 30 s.
For both studies, the thermocycling protocol and the microfluidic processing were accomplished by means of a dedicated compact LabDisk Player instrument ( Figure 4, QIAGEN Lake Constance, Stockach, Germany). The detailed microfluidic protocol is described in Supplementary Table S3. Rotational frequency control and air-heating enabled centrifugo-pneumatic [43,46] and thermo-pneumatic microfluidic unit operations [42,44,47], and allowed for a time of 140-180 min from sample inlet to final result (depending on the Vector or NA Study). PCR raw data acquired with the LabDisk Player were converted and analyzed using the RotorGene Software (QIAGEN, Hilden, Germany).

The Vector Study Results
In the Vector Study, we aimed to demonstrate the simultaneous detection of several DNA and RNA targets in triplex configuration. We tested 18 disks using diluted mosquito lysates as samples, and the arrangement of the assays (primers/probes per reaction chamber) is shown in Table 2.
This work focuses on the semi-quantitative screening of pooled specimens. The usefulness of the initial semi-quantitative screening of pooled mosquito specimens (detection/no detection of a mutation or pathogen) is supported by the two following paradigms. It is not uncommon to screen large numbers of individual mosquitoes and eventually find out that the whole population is wild type for a particular mutation (0% mutant allele frequency) or fixed for another one (100% mutant allele frequency). Furthermore, there are instances where the pathogen (Plasmodium or arbovirus) may not be present at all (low transmission settings) in several study sites, and these can be ruled out from a further, more detailed investigation.
The main goal of a semi-quantitative screening is to distinguish clearly between positive and negative results. This can be achieved by considering the gap between the maximum number of PCR cycles and the highest observed Ct value of a positive result. A maximum number of 40 or 45 PCR cycles (depending on the study) was fixed, according to authors' previous work taken as reference [34]. All targets which were not detected until that cycle number were considered as negative test results. The highest Ct values of positive results were in the range of 33 (Supplementary Table S4). The difference between that number and the maximum number of PCR cycles means that the concentration between the 'weakest' positive and the negative is at least two orders of magnitude. Therefore, we can clearly discriminate between positive and negative values. The RPS7 assay, targeting a house-keeping gene, can be considered as a positive amplification control, as it is expected to be positive in one of the three fluorescent wavelengths (FAM) and in four reaction chambers. To compare this with the assays validated in the preceding assay development work, the authors Mavridis et al. [34] had also used 40 cycles for their PCR (using a ViiA 7 Real-Time PCR System, Applied Biosystems, Waltham, MA USA), and their highest Ct value of positive results was of the order of 34.
In Table 3 we present an overview of the true positive (TP), false negative (FN), true negative (TN), and false positive (FP) rates for each assay and for each mosquito pool, as well as in total for

The Vector Study Results
In the Vector Study, we aimed to demonstrate the simultaneous detection of several DNA and RNA targets in triplex configuration. We tested 18 disks using diluted mosquito lysates as samples, and the arrangement of the assays (primers/probes per reaction chamber) is shown in Table 2.
This work focuses on the semi-quantitative screening of pooled specimens. The usefulness of the initial semi-quantitative screening of pooled mosquito specimens (detection/no detection of a mutation or pathogen) is supported by the two following paradigms. It is not uncommon to screen large numbers of individual mosquitoes and eventually find out that the whole population is wild type for a particular mutation (0% mutant allele frequency) or fixed for another one (100% mutant allele frequency). Furthermore, there are instances where the pathogen (Plasmodium or arbovirus) may not be present at all (low transmission settings) in several study sites, and these can be ruled out from a further, more detailed investigation.
The main goal of a semi-quantitative screening is to distinguish clearly between positive and negative results. This can be achieved by considering the gap between the maximum number of PCR cycles and the highest observed Ct value of a positive result. A maximum number of 40 or 45 PCR cycles (depending on the study) was fixed, according to authors' previous work taken as reference [34]. All targets which were not detected until that cycle number were considered as negative test results. The highest Ct values of positive results were in the range of 33 (Supplementary Table S4). The difference between that number and the maximum number of PCR cycles means that the concentration between the 'weakest' positive and the negative is at least two orders of magnitude. Therefore, we can clearly discriminate between positive and negative values. The RPS7 assay, targeting a house-keeping gene, can be considered as a positive amplification control, as it is expected to be positive in one of the three fluorescent wavelengths (FAM) and in four reaction chambers. To compare this with the assays validated in the preceding assay development work, the authors Mavridis et al. [34] had also used In Table 3 we present an overview of the true positive (TP), false negative (FN), true negative (TN), and false positive (FP) rates for each assay and for each mosquito pool, as well as in total for the Vector Study. The classification was done according to the expected outcomes based on previous work with these laboratory mosquitoes [29,31,34]. Some of the included assays were expected to be negative for the specific laboratory-reared mosquitoes (TN, also included in Table 3). In case a positive-expected assay was not detected, it was attributed to be false negative (FN). No FN results were found for the DNA assays. Notably, we also did not observe FP results in the Vector Study for any assay or in any of the five pools (Table 3), which demonstrates the high specificity of the disk-integrated assays. Some representative real-time PCR curves acquired by a VectorDisk are shown in Figure 5. positive-expected assay was not detected, it was attributed to be false negative (FN). No FN results were found for the DNA assays. Notably, we also did not observe FP results in the Vector Study for any assay or in any of the five pools (Table 3), which demonstrates the high specificity of the diskintegrated assays. Some representative real-time PCR curves acquired by a VectorDisk are shown in Figure 5. Another key output of this study was the detection of twelve RNA assays using a combination of geometric multiplexing (four reaction chambers: 9, 10, 11, 12) and color multiplexing (three wavelengths detecting in each chamber). Data from the assay development (previous work of the coauthors Mavridis et al. [34]) indicate that the triplex versus singleplex RT-PCR does not lead to loss of efficiency. The assays were successfully validated in terms of expression levels against standard two-step singleplex PCR assays and microarrays, using laboratory strains and field-caught samples. Further quantitative characterization of the efficiency, sensitivity, specificity, and reproducibility of the assays themselves is available in the co-authors' previous work [34]. In short, multiplex assays were efficient (reaction efficiencies = 95-109%), sensitive (covering a >10.0 Ct range up to Ct = 33.0 with R 2 values > 0.99), specific (TaqMan chemistry), and reproducible (CV = 4.5-12.1%).
Contrary to the DNA assays, FN results were found among the RNA assays and, mostly, in the red channel. In particular, some of the Detox assays (e.g., Detox (B) K1 in yellow channel, Detox (A) CYP6M2 and Detox (B) CYP6P4 in red channel) exhibited a higher number of FN. The higher FN rate of the RNA assays may be attributed to the fact that RNA amplification includes one additional stepthe RNA reverse transcription-which is more prone to inhibitors than the DNA amplification [48]. One more factor that may account for this higher FN rate in the case of RNA assays could possibly be the adsorption of reverse transcriptase to the disk surface. Given the fact that this enzyme is integrated into the lyophilized pellet that is stored in chamber C (Figure 3), undergoes rehydration and mixing, and the resultant liquid is then distributed to the reaction chambers (E), this appears to Another key output of this study was the detection of twelve RNA assays using a combination of geometric multiplexing (four reaction chambers: 9, 10, 11, 12) and color multiplexing (three wavelengths detecting in each chamber). Data from the assay development (previous work of the co-authors Mavridis et al. [34]) indicate that the triplex versus singleplex RT-PCR does not lead to loss of efficiency. The assays were successfully validated in terms of expression levels against standard two-step singleplex PCR assays and microarrays, using laboratory strains and field-caught samples. Further quantitative characterization of the efficiency, sensitivity, specificity, and reproducibility of the assays themselves is available in the co-authors' previous work [34]. In short, multiplex assays were efficient (reaction efficiencies = 95-109%), sensitive (covering a >10.0 Ct range up to Ct = 33.0 with R 2 values > 0.99), specific (TaqMan chemistry), and reproducible (CV = 4.5-12.1%). Table 3. Overview of the true positive (TP), false negative (FN), true negative (TN), and false positive (FP) rates for each assay and for each mosquito pool, as well as in total for the Vector Study. The five different colors (Pool #1 . . . #5) refer to the five different pools of mosquitoes (Figure 1). The numbering in the first column indicates the VectorDisk reaction chamber where an assay is expected positive or negative. The grey horizontal shading in some assay rows (accompanied by the symbol '-') means that neither positive nor negative signal was expected, because the primer/probe mixtures stored in these chambers were expected to give signals in other detection channels. Contrary to the DNA assays, FN results were found among the RNA assays and, mostly, in the red channel. In particular, some of the Detox assays (e.g., Detox (B) K1 in yellow channel, Detox (A) CYP6M2 and Detox (B) CYP6P4 in red channel) exhibited a higher number of FN. The higher FN rate of the RNA assays may be attributed to the fact that RNA amplification includes one additional step-the RNA reverse transcription-which is more prone to inhibitors than the DNA amplification [48]. One more factor that may account for this higher FN rate in the case of RNA assays could possibly be the adsorption of reverse transcriptase to the disk surface. Given the fact that this enzyme is integrated into the lyophilized pellet that is stored in chamber C (Figure 3), undergoes rehydration and mixing, and the resultant liquid is then distributed to the reaction chambers (E), this appears to be an explanation. The above options are recorded as candidate investigation/optimization steps for the integration of the RNA-based insecticide resistance assays.
For an assessment of the intra-pool reproducibility of the results, we conducted a basic statistical analysis for each pool individually, which is summarized in Supplementary Table S4. Furthermore, and in order to have an indication of the inter-pool variation, we performed a weighted averaging (grey-colored 'Overall' columns of Supplementary Table S4), because for each specific assay we did not have the same number of data points (due to some FN cases). In an inter-pool comparison, the calculated CV of each assay was below 8% (the CV of the DNA assays ranging from 1.8% to 4.1%, while the CV of the RNA assays ranging from 3.2% to 7.9%). The CVs of individual assays in five intra-pool comparisons were also of this range. The main factors that may drive the inter-pool variation are the manual steps in disk preparation (pipetting of primers/probes, drying in the oven), and the manual preparation of mosquito samples, including the crude lysis. Therefore, the low variability-even between pools-can be attributed to the automated aliquoting into preloaded reaction cavities and is a strong indicator of the robust and reproducible performance of the VectorDisk. Regarding the intra-disk microfluidic reproducibility (i.e., among the reaction chambers), one could also make an assessment from the very comparable Ct values of the assay RPS7, targeting a house-keeping gene, which acts as normalizer, and which is detected in four chambers (9, 10, 11, and 12) in the green detection channel (Supplementary Table S4).
Overall, the Vector Study demonstrated the compatibility of the LabDisk platform with the crude lysis-direct amplification process. Results are in line with past work on assay development by the co-authors Mavridis et al. [34], who have shown that the crude lysis-direct amplification option performed very closely to a typical magnetic bead-based bind-wash-elute extraction and purification followed by PCR (comparison performed by means of the resulting Ct values). Furthermore, previous work on microfluidic integration by the authors has demonstrated the possibility of homogenizing biological matrices (e.g., human saliva [49], bacteria [50], cells [51], swabs [52]) using disk-integrated magnets that are actuated by external (still on LabDisk Player) magnets [53]. Therefore, upon suitable adaptation of the VectorDisk microfluidic design, the lysis and homogenization of mosquito pools could be translated from an ex situ manual (currently) process to an in situ automated (on disk) process, and be followed by in situ dilution and amplification towards a truly fully-automated vector-to-result analysis. This could offer an even higher degree of automation, and result in less overall analysis time, costs, and potential errors than performing manual mosquito processing and multiple PCR assays.

The Nucleic Acid Study Results
In the NA Study, the authors aimed to explore the applicability of the VectorDisk in the field of arboviral vectors in terms of the performance and reproducibility of the corresponding assays integrated in the disk. As no arboviral vectors were available, the experiments were done using nucleic acids (WNV-Lineage 1 and ZIKV). Furthermore, given that the mosquitoes used in the Vector Study were laboratory-reared and did not contain any malaria parasites, purified DNA and RNA from infected mosquitoes were used in the NA Study (sample 'PF-INF'). The purification process is described in previous work [29]. Nine disks were used, and the arrangement of the assays (primers/probes per reaction chamber) is shown in Table 2. From the results, we extracted true/false positivity/negativity rates, for each assay and in total, which are shown in Table 4. Each disk with a viral target was expected to give TP signals from two chambers where the corresponding primers and probes were pre-stored, and TN signals from all other eight chambers. Each disk with the sample mixture 'PF-INF' was expected to give TP signals from four chambers (#5 and 10 for DNA, and #6 and 11 for RNA detection), and TN signals from all other six chambers. This was indeed the case, and no FPs were observed, which indicates an excellent performance of specificity of the disk-integrated arboviral assays. Some representative real-time PCR curves are shown in Figure 6. The disks also included primer/probes for detection of dengue virus (DENV T1-4), WNV-Lineage 2, and P. ovale/vivax/malariae ( Table 2), but since the corresponding targets were not present in any of the samples, only TN or FP could be expected from these assays. Therefore, these primers/probes were used for cross-specificity purposes (tested against the other samples) and notably, only TN results were extracted. Furthermore, after analyzing the data for a reproducibility assessment, it is noteworthy that all assays in the VectorDisk performed with an inter-disk CV < 6% (Supplementary Table S5). require no cold chain for transport, which is essential when the target settings are tropical regions. A comprehensive review of candidate analytical methods and platforms has been published by Vontas et al. [16]. The fact that the LabDisk platform has been shown to successfully detect tropical infections on human samples (malaria, dengue, chikungunya) [26,54,55], in combination with the current demonstration of compatibility with analysis of multiple vector-related assays, paves the way for a One Health approach of combined human/vector diagnostics [22].   TP  FN  TN  FP  TP  FN  TN  FP  TP  FN  TN  FP  TP  FN  TN  FP  2 WNV-Lineage  TP  FN  TN  FP  TP  FN  TN  FP  TP  FN  TN  FP  TP  FN  TN  FP  2 WNV-Lineage 2 3/3 0/3 3/3 0/3 3/3 0/3 9/9 0/9 3 -4 -5 Plasmodium ovm 1 3/3 0/3 3/3 0/3 3/3 0/3 9/9 0/9 6 -7 WNV-Lineage 2 3/3 0/3 3/3 0/3 3/3 0/3 9/9 0/9 8 -9 -10 Plasmodium ovm 1 3/3 0/3 3/3 0/3 3/3 0/3 9/9 0/9 11 -Red detection channel  TP  FN  TN  FP  TP  FN  TN  FP  TP  FN  TN  FP  TP  FN  TN  FP  2  -3  ZIKV  3 Overall, the NA Study was performed by using the same disk design and fluidic operations, as well as the same lyopellet as the Vector Study, by only exchanging the primer/probes in the reaction chambers, and by slightly modifying the PCR protocol. This easy adaptability to the needs of the analysis is of utmost importance for an automated system, as it allows for a higher volume of manufacturing, resulting in lower costs (several different requests can be served by the same disk design). Furthermore, such a configuration allows a fast response to urgent needs-for instance, in the case of epidemics-because the primers/probes are de-coupled from the amplification reagents. Also important is the fact that the reagents have been developed and integrated in a way that they require no cold chain for transport, which is essential when the target settings are tropical regions. A comprehensive review of candidate analytical methods and platforms has been published by Vontas et al. [16]. The fact that the LabDisk platform has been shown to successfully detect tropical infections on human samples (malaria, dengue, chikungunya) [26,54,55], in combination with the current demonstration of compatibility with analysis of multiple vector-related assays, paves the way for a One Health approach of combined human/vector diagnostics [22].

Conclusions
This work included two proof-of-principle studies. The Vector Study demonstrated the capacity of the VectorDisk to simultaneously and semi-quantitatively detect DNA-and RNA-specific targets in multiplex assays from mosquito pool samples, which were crudely lysed, diluted, and then directly amplified in the disk. A combination of multiple microfluidic reaction chambers and detection wavelengths (geometric and color multiplexing) was successfully used for this scope. The NA Study, starting from purified nucleic acids, demonstrated the rapid adaptability of the platform by changing only the primer/probe panel and the capacity to detect arboviral vector and malaria-infected vector assays. In both studies, key targets relevant to species identification, infective stage, and insecticide resistance markers, were detected. Both studies indicated a high reproducibility in detection of the assays, with intra-and inter-pool CVs of the PCR Ct values being <10%. High specificity was also achieved with no FPs detected in either of the Vector or NA Studies.
Overall, the outcomes of our work give a promising perspective on the platform to be applied in fully automated vector diagnostics-a platform which, once tested and validated with field-collected disease vectors, will contribute molecular entomological data that is highly relevant to decision-making in vector control programs. Regarding this implementation, we foresee two further steps: (i) the in situ (on-disk) lysis and homogenization of mosquitoes that will enable fully integrated sample preparation, which can be advantageous for minimizing hands-on work; and (ii) the transition from semi-quantitative to quantitative analysis, which will offer more detailed information related to insecticide resistances, contributing to vector surveillance and control.
Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9717/8/12/1677/s1, Table S1: Primers/probes sequences for the assays in the Vector Study, Table S2: Primers/probes sequences for the assays in the NA Study, Table S3: Microfluidic protocol to operate the VectorDisk, Table S4: Statistical analysis of the Ct values derived from five pools of mosquitoes tested with the VectorDisk during the Vector Study, Table S5: Statistical analysis of the Ct values derived from the VectorDisk results from the NA Study.