An integrated versatile lab-on-a-chip platform for the isolation and nucleic acid-based detection of pathogens

Aim: Processing of the samples in molecular diagnostics is complex and labor-intensive. An integrated and automated platform for sample preparation and nucleic acid-based detection can significantly relieve this burden for the users. Results: We present a prototype of a versatile and integrated platform for the detection of pathogens in various liquid media. We describe a proof-of-concept for the integrated isolation of bacteria, cell lysis with optional DNA extraction, DNA amplification and detection in two different reactions, loop-mediated isothermal amplification and PCR, on a single microfluidic platform. Conclusion: The platform enables the transition from large sample volume to microfluidic format. The design and open interface enable its versatile application for various nucleic acid-based assays, from simple to complex setups.

equipment and facilities. Apart from that, application of DNA amplification techniques to a real sample may be hindered by interfering compounds and inhibitors, in particular when the concentration of target cells is low. For instance, diagnostics of infectious diseases is often associated with the presence of a few micro organisms in a large volume of complex medium: blood, urine, cerebrospinal fluid, etc. [1][2][3]. In this case, additional sample preparation is required for the iso lation and concentration of the analyte. In diagnostic practice, the workload of the personnel often does not allow for the individual manual processing of challeng ing samples, although the result of the sample analysis is urgent, in particular in life threatening infections. Conventional labautomation solutions, for example DNA extraction stations, are usually designed for the high throughput processing, and their application to individual samples is inexpedient. In such situations, the utilization of the labonachip (LOC) device leading to an accurate and fast detection of a disease causing pathogen can be an optimal strategy.
The concept of LOC pursues the development of a sampletoresult device, which allows for the inte gration of complex processing steps on an automated platform. Multiple systems for DNA amplification on a chip have been reported [4][5][6][7][8][9]; however, most of these devices are able to process only small sample volumes (usually ≤100 μl). In the applied analytics, it is often necessary to process a significantly higher sample vol ume in order to obtain a detectable number of patho gens. Also, LOCs aiming at integrated DNA extrac tion, amplification and detection frequently have an utterly complex structure for the control of cartridge components (pumps, valves), which increases the cost of such systems.
Here we describe a prototype of a LOC platform for the detection of infectious agents in various liquid media. The platform is designed to provide several fea tures: transition from a large sample volume (≥1 ml, highly scalable) to a microfluidic format; integration of pathogen isolation, lysis, DNA extraction and ampli fication, and real time detection; simplicity of micro fluidics; and flexibility. Different NAT assays can be accommodated on the platform. Our system includes a microfluidic cartridge and an external tabletop instru ment. A simple linear design of the cartridge is based on the stationary microfluidics, which does not require valves or actuators [10][11][12][13][14][15]. This concept is realized via the implementation of functionalized magnetic beads for pathogen isolation and DNA extraction. The exter nal instrument maintains the bead transportation and temperature control in the reaction chambers in a userindependent mode, and the userfriendly soft ware allows adjusting these processes to the various needs. The detection of the amplified product is per formed with a LEDbased optical unit that provides the readout of the fluorescent signal. In the current work, we demonstrate the proofofconcept for the detection of E. coli and Salmonella bacterial species as two model pathogens in the spiked liquid samples via loop mediated isothermal amplification (LAMP) and PCR. We worked with 1 ml samples in our demon stration experiments: this volume can be easily scaled up; however, an integration of an additional magnet on the lateral side of the sample container is considered to improve the recollection of the beads from the large volumes.

Cartridge design & fabrication
The cartridges (called MinoCards within the related project) were designed and produced by the company microfluidic ChipShop (Germany) by injection mold ing of polycarbonate. The bottom side of the cartridge was bonded with a 175 μmthick polycarbonate foil. The cartridge comprised three identical lanes for simultaneous processing of three samples; reaction chambers are designated in Figure 1.
The MinoCard can accommodate assays of various complexities. In a simple assay, lysis, DNA amplifica tion and realtime detection are conducted in chamber 7, while the preceding chambers are used as washing chambers. In a more complex assay, pathogen lysis may be performed in compartment 4 outside of the amplification chamber, while DNA can be transferred using magnetic beads to the latter through the wash ing chambers 5 and 6. For the most complex multiplex assay, a hybridization chamber 9 for a microarray chip is provisioned. Here amplified nucleic acid products can be hybridized to complement oligonucleotides immobilized on a magnetoresistive chip. The reagents can be preserved in the reaction chamber in a lyophi lized form as a dry pellet. They will be added in liquid form on the stage of the manufacturing of the car tridges and lyophilized in situ. The cartridge will be sealed afterwards. A buffer or water for the resuspen sion can be provided in a sealed blister mounted onto position 1.
In the current publication, we present simple NAT assays with LAMP and PCRbased detection, and a more complex assay that also comprises DNA purification.

The external instrument
The external tabletop instrument (MinoLyzer) was constructed in compliance with the design of the microfluidic cartridges. It includes movable electro magnets for the transport and steering of magnetic An integrated versatile lab-on-a-chip platform for the isolation & nucleic acid-based detection of pathogens Research Article future science group beads, a heating module, which contains four indepen dent Peltier elements, and an optical detection module ( Figure 2B). Magnetic transport of the beads involves a sequence of the following steps: move the electro magnet to the position where the beads must be col lected, turn on the electromagnet, allow the collection of the beads, slowly move the working electromagnet along the Xaxis, stop and turn off the magnet, and allow spontaneous resuspending of the beads. A user can program the appropriate processing parameters for the magnetic bead steering, such as the position of the magnet at the defined time point, the velocity of its movement along the horizontal axis, and the power of the magnetic field.
Once the program is stored on a SDCard and inserted into the instrument, all steps are automati cally controlled with minimal manual intervention. The Peltier elements allow the controlled heating of the areas T1T4 of the cartridge over a desired period of time ( Figure 2A). The temperature during the heating steps is measured via the Negative Temperature Coef ficient microsensors integrated in the copper surfaces of the heating elements. The copper surfaces also sup port an even heat transfer into the reaction chambers of the microfluidic cartridge. A proportional-integralderivative controller realizes heating or cooling of the Peltier elements.

Preparation of the cartridges for the assay
The inner surface of the amplification chamber was saturated with 1% (w/v, in water) bovine serum albu min (BSA, molecular biology grade, Carl Roth, Ger many) for 30 min. Subsequently, 50 μl of 0.12% (w/v) BSA + 0.4% (v/v) Tween 20 (Carl Roth, Germany) was boiled in the PCR chamber for 5 min at 95°C. The remaining liquid was removed, and the top side of the MinoCard was sealed with the transparent qPCR foil (either Nerbe, Germany, or Roche, Germany). While a common surface preparation usually exploits satura tion with BSA, we used additional detergent and heat ing to inactivate and/or remove any chemical inhibi tors associated with the plastic chip material or foil. The entire lane (ca. 200 μl) was then filled with reac tion mix (Table 1). Finally, the sample container was attached to the socket, and the cartridge was placed onto the instrument.
For the fully integrated assay, beads and reagents for DNA extraction were deposited as follows and air dried prior to sealing the cartridge: chamber 3 (refer to

Isolation of bacteria from liquid sample
Salmonella enterica (serovar enteriditis) or Escherichia coli DH5α were grown overnight in LB medium at 37°C, pelleted by 5 min centrifugation at 8000 × g and resuspended in phosphate buffered saline (PBS, pH 7.4). Approximate quantification of bacteria was done by optical density measurement at 600 nm.
Magnetic nanoparticles for the capture of the bac teria were prepared from MagPrep ® P25 carboxylated beads (Merck, Germany). One mg of beads was acti vated by 30 min incubation with conjugation reagents (Sigma Aldrich, Germany): 50 μl of 1ethyl3(3 dimethylaminopropyl)carbodiimide (50 mg/ml in cold 100 mM morpholinoethanesulfonic acid (MES), pH 5.3) and 50 μl of Nhydroxysuccinimide (50 mg/ ml in cold 100 mM MES). The particles were washed with 25 mM MES + 0.05% (v/v) Tween 20 followed by a washing step in PBS + 0.05% Tween 20 (PBST). The beads were resuspended in 100 μl of PBST, and 50 μl of protein A (1 mg/ml in PBS) was added. The conjugation was carried out at room temperature with constant shaking for 2 h. The supernatant was removed and replaced with 100 μl 1 M Tris (Sigma Aldrich, Germany) for 30 min to saturate the remaining acti vated carboxyl groups. The beads were washed two times with PBST and resuspended in PBST to the final concentration of beads 10 mg/ml.
A quantity of 5 μg of antiSalmonella antibod ies (Acris Antibodies, Germany, catalogue No. AM03096PUN) or antiE. coli antibodies (catalog No. AM00717PUN) was added to the beads, and the suspension was incubated for 30 min at room tem perature. Unbound antibodies were removed by three washing steps with PBST.
A quantity of 1 ml of PBS was spiked with approxi mately 5 × 10 8 S. enteritidis colony forming units (CFU)/ml or approximately 1 × 10 8 E. coli CFU/ml. A quantity of 1 ml of sterile PBS was used as a negative control. A quantity of 50 μg of antibodyconjugated magnetic beads (Abbeads) was added to the samples and incubated for 30 min at room temperature on a rotating mixer.
The samples were then transferred to the cartridge that was placed into the MinoLyzer instrument. The  beads were recollected with a magnet and magnetically transported to the lane of the card loaded with liquid reaction mix. The reference samples were analyzed using conventional equipment and reaction setup: thermocyclers and PCR vials or plates. The current article is focused on the spiked buffer samples as demonstrators. We also performed a limited number of experiments to evaluate the compatibility of the system with whole blood. Generally, it is nec essary to apply a stronger magnetic field for a longer time in comparison to buffer samples for the collection of the particles and/or perform the collection in a few repeated steps. These parameters must be optimized for the different matrices and sample volumes. These experiments are a subject of the future investigations.

Pathogen lysis & DNA amplification on the cartridge
We evaluated the detection of Salmonella with LAMP and E. coli with PCR. The beads with the captured pathogen were magnetically transferred through the washing chambers straight to the amplification cham ber. The Peltier element T3 (refer to Figure 1) was programmed to an appropriate temperature regime.
For LAMP, the chamber was heated to 65°C for 35 min to maintain isothermal DNA amplification. It was empirically determined that this heating program also enables the lysis of Salmonella. The presence of the magnetic beads during the amplification was found to have no major adverse effect on the robust LAMP reaction.
For PCR, the chamber was initially heated to 95°C for 5 min to lyse E. coli. After the lysis, the unloaded Abbeads were magnetically transferred backward to the cavity 6. The PCR was performed according to the program: 3 min initial denaturation at 95°C fol lowed by 40 cycles of 25 s denaturation at 95°C, 25 s annealing at 60°C and 25 s elongation at 72°C. Final elongation for 1 min at 72°C completed the program.
The detection was performed in a real time mode with the fluorescence detector ESElog (Qiagen, Ger many): excitation 470 nm/emission 520 nm. The acquisition of the signal was done every minute for LAMP or in each elongation phase for PCR.

Pathogen lysis & DNA amplification in reference samples
For Salmonella LAMP reference, the beads with the captured bacteria were washed twice in 100 μl PBST, 24 μl of the LAMP reaction mix was added, and the reaction was performed in a LightCycler ® 480 Real Time PCR System (Roche, Germany) for 35 min at 63°C. A melting curve was created at the end of amplification to verify the specificity of the products.
For E. coli PCR reference, the beads were washed in the same manner, resuspended in 9 μl water and heated to 95°C for 5 min for the lysis of the pathogen. A quantity of 8 μl of the lysate without the Abbeads was mixed with 12 μl PCR reaction mix and processed in TProfessional Thermocycler (Biometra, Germany). The samples were analyzed by electrophoresis in 1.5% agarose gel with subsequent ethidium bromide staining.

Fully integrated LOC detection of E. coli
Fully integrated assay comprised immunomagnetic capture of E. coli cells, their magnetic transfer to the cartridge, pathogen lysis, DNA extraction, amplifica tion in PCR and a realtime detection on the Mino Card and MinoLyzer. The MinoCard was prepared for the full assay version and positioned on the Mino Lyzer. The Abbeads with the captured bacteria were magnetically transferred to the chamber 4. The lysis of the captured pathogens was done in chamber 4 using Peltier element T1 at 95°C for 5 min. The Abbeads were then transferred backward toward the inlet and rested there, since they have no further function after the pathogen release and lysis.  The DNAbinding CST beads were transferred from their position in the chamber 6 to chamber 4 and incu bated there with the bacteria lysate for 5 min at room temperature for the DNA binding. During the incuba tion, the beads were magnetically agitated (collected and released) for 5 s every minute for sample mixing. Then the beads were transferred to chambers 5 and 6 subse quently for short washing steps (1-2 resuspension/recol lection cycles) and dragged to the PCR chamber 7. There, DNA elution was done at 65°C for 5 min and 95°C for 1 min with the bead agitation for 5 s every minute. PCR mix has a pH of 8.5 and thus acts as elution buffer in this system. After the DNA elution, CST beads were trans ported backwards to chamber 5, and E. colispecific PCR was performed employing underlying Peltier element T3 with a continuous monitoring of fluorescence.
Reference samples for the fully integrated assay were processed manually in the vials. Abbeads after the pathogen capture were incubated with 20 μl 0.5 M binding buffer, and the lysis was supported by heat ing to 95°C for 5 min. The supernatant without the Abbeads was mixed with 50 μg CST beads and incu bated for 5 min at room temperature. After two wash ing steps in 10 μl PCR buffer + 1 μl 0.25 M binding buffer, DNA was eluted into 20 μl PCR mix, and PCR was performed in LightCycler.

Results & discussion
In the described work, we focus on the proofofcon cept demonstrations of the functional capabilities of the platform and do not provide quantitative investi gations of the sensitivities or specificities of the shown assays. We realize that the readers would be interested in the limits of detection (LOD) of the platform. By the moment we discovered the need for certain adjust ments in the platform parts that we describe in this section. We aim to perform the investigation of the LOD after these improvements and hope to satisfy the interest of the readership in a followup article.

Lab-on-a-chip detection of S. enteritidis via LAMP
We demonstrated a reproducible effective detection of Salmonella following its immunomagnetic isolation and isothermal DNA amplification in the MinoLyzer. We could observe a prominent increase of the fluores cence signal after 15-16 min of amplification (Figure 3) in the positive samples, while in the negative controls the fluorescence remained at the base level for the entire period (35 min).
In these experiments, we used a high concentration of bacteria (5 × 10 8 CFU/ml) for a proofofconcept demonstration case. Diagnostically relevant pathogen loads are much lower and will require longer amplifica tion times. Although a user can easily program a longer amplification period, a current issue in this scope is the specificity of the fluorescent signal acquired at the late stages of LAMP. This issue could be easily resolved in the LightCycler by melting curve analysis; yet the cur rent version of our platform did not allow an accurate recording of the melting curve. Therefore, here we restricted our amplification program to 35 min, which enabled an unambiguous interpretation of the results.
It is necessary to mention that we avoided verifi cation of the LAMP results in gel electrophoresis. According to our experience, LAMP is exquisitely sus ceptible to carryover contamination with the products of amplification. We strongly advise avoiding any post amplification manipulations with the LAMP vials or cartridges, in order to minimize the probability of the false positive results. A fully integrated detection is, in any case, an absolute requirement for a LOC device, thus improvements in regards to higher specificity will be needed for the future versions of the assay.

Lab-on-a-chip detection of E. coli via PCR
In order to demonstrate the ability of the platform to accommodate different NAT assays, we investigated the performance of the integrated PCR on the Mino Card. In comparison to the isothermal amplifica tion described in the previous section, PCR involves a significantly more complex heating regime with the periodically changed temperatures. Another differ ence between PCR and LAMP is the need for the high temperature steps: 95°C for DNA denaturation. We evaluated the possibility to maintain this reaction with the MinoLyzer and checked its compatibility with the linear valvefree design of the MinoCard.
We were able to obtain positive PCR results using the program parameters similar to a conventional ther mocycler and to complete 40 PCR cycles on the Mino Lyzer within approximately 1.5 h. We could detect E. coli after its immunomagnetic preconcentration from the model samples with 10 8 CFU/ml and achieved a clear differentiation between the positive and nega tive samples (Figure 4). The samples were characterized by an average Cq 28.0 ± 0.6 (n = 3) ( Figure 4A). Thus, we generally demonstrated the technical capability of the platform to perform complex NAT assays. Never theless, we faced a few hindrances while obtaining and analyzing the fluorescent signal.
As it is evident in Figure 4, the shape of the amplifi cation curves was affected by the signal fluctuation at random points. This is mainly caused by the eventual formation of the small bubbles in the PCR chamber during the heating to high temperatures, which led to a scatter of fluorescence. It is especially salient for the curve S3 in Figure 4A. For this sample, we used a www.future-science.com 10.4155/fsoa-2016-0088 www.future-science.com future science group future science group An integrated versatile lab-on-a-chip platform for the isolation & nucleic acid-based detection of pathogens Research Article future science group running average function to smoothen the data, since the overall exponential growth of the fluorescent signal was unquestionably visible. It is likely that the simi lar local fluidic disturbance affected the signal in the negative control. Formally, the negative control is also assigned with a Cq 35.7; however, the curve did not demonstrate an exponential increase of the signal. It was credited as negative after the analysis of the plot, which was further confirmed by gel electrophoresis ( Figure 4B).
In Figure 4B, a formation of some side amplification products is noticeable, which are likely to be primer dimers and remaining primers. The latter is strongly undesired in the realtime PCR with an unspecific  intercalating dye such as SYBRGreen ® which is used here, thus an optimized PCR demanded for the plat form, preferably probebased to improve the specificity of the detection. A significant factor, which strongly affected the PCR efficiency and reproducibility in the MinoCard, was the design of the amplification chamber. As it is displayed in Figure 1A, the chamber is connected with the adjacent compartments by thin channels. In a theoretical model, the diffusion of the liquid between the connected compartments is negligible, which is generally confirmed by the successful amplification of nucleic acids in the given structure. However, dur ing the PCR, it is immensely increased: the eventu ally occurring air bubbles expand and collapse along with the heating/cooling cycles, and create therefore a pumping effect. Due to this, larger portions of liq uid may be extruded from the amplification chamber into the adjacent cavities. As a consequence, the PCR yield decreases. The implementation of valves in the amplification chamber will provide consistency of the physical and chemical conditions during the ampli fication leading therefore to a reproducible PCR performance.

Fully integrated detection of E. coli via PCR
In the full version of the assay, we pursued the inte gration of such complex steps as pathogen precon centration, lysis, DNA purification, amplification and detection on a very simple linear chip with sta  An integrated versatile lab-on-a-chip platform for the isolation & nucleic acid-based detection of pathogens Research Article future science group tionary microfluidics. In the simple assay, we have already proven the capability of the platform for some of these processes; however, the implementa tion of DNA purification is one of the most challeng ing procedures. Extraction of DNA requires a series of successively changing reagents and conditions, where the first step is typically binding of nucleic acids to a solid carrier. In our system, we also exploit magnetic beads for this purpose. A conventional magnetic beadbased DNA purification utilizes silica particles that bind DNA in the presence of high con centration of chaotropic salt. This agent is known as a strong PCR inhibitor; therefore, this method is not suitable for the valvefree system due to an unavoid able minor carryover of the salt to the PCR chamber. Thus, we chose a beadbased method which imple ments mild medium for DNA binding. CST beads capture DNA at pH <6.5; washing of the particles is performed at pH ∼7.0 and elution of nucleic acids is effective at pH 8.5, which corresponds to PCR con ditions [19].
We used a custom binding buffer based on potas sium acetate, pH 4.4, in order to create the required pH profile in the chip ( Figure 5). Since the lane of the chip is filled with PCR mix with pH 8.5, we used an accu rate dosage of the binding buffer which provided the necessary conditions for DNA binding and washing steps with the consideration of the interaction between the buffers. At the same time, an eventual carryover of the binding buffer to the PCR chamber should not have a significant effect on the PCR performance: its minor amounts are neutralized by a substantially larger volume of PCR buffer. Also, potassium acetate per se does not pertain to PCR inhibitors, in the contrast to any chaotropic salts.
We obtained signals characterized by Cq = 32.1 ± 0.5 (n = 3) for the samples F1F3 processed on the MinoLyzer ( Figure 6A); the efficiency of detection was generally lower than on the LightCycler (Cq = 26.8 for the reference sample). Further analysis of the samples by gel electrophoresis revealed that no specific prod uct was generated in the sample F3, and the fluores cence was generated by unspecific side product (primer dimers; Figure 6B). For the samples F1 and F2, distinct specific bands were observed on the gel. Thus, we faced the issue of the specificity in optical detection of the amplified products. The problem itself is known for SYBRGreen ® based detection, since this unspecific intercalator produces a signal with any doublestranded DNA. Performing PCR with the sequencespecific labeled probes would significantly improve the speci ficity of the assay. Optimization of PCR in order to reduce the generation of unspecific products will also have a positive impact. Finally, in the given settings, an analysis of melting temperatures of the amplicons can be recommended. We attempted an acquisition of a melting curve on the MinoLyzer using the available optical module (data not shown); however, an accurate detection of the fluorescent signal at the high tempera tures was often hindered by the random formation of the air bubbles in the area of detection. Thus, a clear melting curve resolution on our platform remains to be validated.
Nonetheless, we have demonstrated that integra tion of quite complex assay protocols can be realized in the simple valvefree system with stationary micro fluidics. There are integrated platforms for molecular detection of the pathogens reported in the literature that are at a higher technology readiness level; how ever, we noticed that none of them combines all of the options available in our system. Thus, there are some platforms based on centrifugal microfluid ics [20,21] that enable automated processing of the samples. Their microfluidic design is fairly complex, while the main limitation is the low sample volume that can be processed: For example, 200 μl of serum (with offchip plasma separation step) in [20] and only 3 μl in [21]. These capacities will be insufficient for the analysis of the samples with the pathogen loads of approximately 10 CFU/ml. Here we would like to underline that we cannot claim the LODs of our plat form at the moment: we revealed and described some present obstacles in the fully integrated assay and see technical strategies for their elimination and improve ment of the assay performance. Subsequently, quanti tative evaluations of the analytical characteristics of the platform are needed.

Conclusion
We developed a functional prototype of a LOC plat form for the integrated NAT detection of the patho gens. We illustrated the versatility of the platform through the demonstration of different amplifica tionbased assays: isothermal reaction (LAMP) and a more complex PCR, using a realtime optical detec tion for two model pathogens. We designed a multi functional microfluidic cartridge that allows a simple transition from large sample volumes to a μl scale, thus enabling preconcentration of an analyte. The socalled MinoCard provisions the accommodation of the NAT assays of various complexity using the same design of the cartridge. This can be realized due to a number of the compartments that can play dif ferent roles in a variety of assays, yet being arranged in a simple linear structure. The latter feature con tributes significantly to the automation of different steps using magnetic beads and the MinoLyzer for their flexible steering according to the assay require ments. We demonstrated this in the complex fully integrated assay that comprised all sample prepara tion steps including pathogen preconcentration and DNA purification in combination with PCR and realtime detection. To our knowledge, this is the first described integration of such a complex assay into the simplest microfluidic design without valves or actuators.   Magnetic beadbased stationary microfluidics obvi ates the need for the integrated liquid actuators in the system, thus allowing for a simpler and cheaper produc tion of the cartridges. Nevertheless, we revealed that an absolutely valvefree system works suboptimally for complex reactions such as PCR. Here we currently see a need for the integration of the valves into the amplifi cation chamber in order to improve the consistency of the inchamber conditions. Yet for the simpler assays such as LAMP, a valvefree design is acceptable. The multifunctional design of the MinoCard and the flex ible operation of the MinoLyzer form a platform with a broad range of capabilities. Here we demonstrated its proofofconcept applications with some common techniques and artificial model samples. Hence, in the future, we see a room for the optimization and broad ening of the platform applications. In addition to the abovementioned adjustment of the MinoCard design, we are striving to optimize the specificity and sensitiv ity of the assays and to evaluate the performance of the platform with clinically relevant pathogen loads. Further development can therefore enable multiplex pathogen detection.
The optimization and validation of the improve ments in the described platform will allow it to serve as a tool for the nucleic acidbased detection of various targets in liquid samples. Preloading the cartridges with the customized reagents in the lyophilized form would allow creation of a palette for the specific assays for the needs of a particular laboratory, which can be processed using the same hardware and fluid ics -the main advantage of a platformbased diag nostics. It is worth mentioning that nowadays more equipmentfree (e.g., smartphonebased) solutions for pointofcare testing actively arise in this field [22][23][24][25]. Nevertheless, it is necessary to understand that the applicability of the smartphonebased assays is limited in the cases where complex sample preparation and heating regimes are required. While the smartphones can enable an optical end point readout, continuous measurements such as real time fluorescence acquisi tion may be also less feasible. Moreover, diagnostic tests supported by personal electronic devices may raise the question of quality control and standardiza tion. Hence, for many diagnostic applications the use of the special instrumentation is unavoidable in the observable future. The aim of our research & develop ment activities is to provide the maximum functions of the platform while keeping the design relatively simple. Such system can be useful in any settings suit able for tabletop instruments, while it obviates the need for the laboratory itself and needs only minimal introductive training.

Future perspective
Making a multifunctional platform fully automated and capable of the processing of various sample matri ces even with low pathogen load is essential for its intro duction into practice. We plan a series of experiments addressing the aspects of the sensitivity of the platform in real world samples, evaluation of various sample vol umes, as well as the assessment and improvement of the timetoresult in these cases. We hope to satisfy the interest of the readers in these details in the followup publications. Once the diagnostically relevant crite ria are fulfilled, equipping healthcare institutions and laboratories with platformbased LOC instruments can provide prompt analysis of individual samples at the point of need and thus support decision making in clinical diagnostics or other fields of applied analytics. Such systems can enable complex molecular analysis of the samples in the settings or situations where this was initially impossible, thus improving quality and speed in the diagnosis of livethreatening pathogens, result ing in hospitalization cost savings and last but not least reducing mortality.