Parallelized Impedance-Based Platform for Continuous Dose-Response Characterization of Antischistosomal Drugs

Abstract

Schistosomiasis is an acute and chronic disease caused by tropical parasitic worms of the genus Schistosoma, which parasitizes annually over 200 million people worldwide. Screening of antischistosomal compounds is hampered by the low throughput and potential subjectivity of the visual evaluation of the parasite phenotypes, which affects the current drug assays. Here, an impedance-based platform, capable of assessing the viability of Schistosoma mansoni schistosomula exposed to drugs, is presented. This automated and parallelized platform enables unbiased and continuous measurements of dose–response relationships for more than 48 h. The platform performance is established by exposure of schistosomula to three test compounds, praziquantel, oxethazaine, and mefloquine, which are known to affect the larvae phenotypes. The system is thereafter used to investigate the response of schistosomula to methiothepine, an antipsychotic compound, which causes complex drug-induced effects. Continuous monitoring of the parasites reveals transient behavioral phenotypes and allows for extracting temporal characteristics of dose–response curves, which are essential for selecting drugs that feature high activity and fast kinetics of action. These measurements demonstrate that impedance-based detection provides a wealth of information for the in vitro characterization of candidate antischistosomals and, represents a promising tool for the identification of new lead compounds.

1. Introduction

Efficient and reliable screening methods are an essential tool for the discovery of novel drug candidates. The demand for improving the identification of promising compounds is particularly high in the field of neglected tropical diseases (NTDs), where funding and commercial investments are typically low.^[1,2] Among the frequently occurring NTDs, schistosomiasis is a tropical disease caused by worms of the genus Schistosoma, which infect over 200 million people worldwide, predominantly children living in poor rural areas of Sub-Saharan Africa.^[3,4] If left untreated, the infection slowly develops into a debilitating chronic disease, which leads to fibrosis of the liver, intestines and/or bladder, anemia, urogenital cancers and, eventually, death.^[4] Despite the high prevalence of schistosomiasis, the current anthelmintic drug discovery pipeline is alarmingly unproductive.^[5] For over 40 years, praziquantel has been the treatment of choice for schistosomiasis and has been widely used as chemotherapeutic agent in mass drug-administration campaigns.^[3] Hundreds of millions of treatments are administered to children and populations at risk each year,^[6] raising a significant potential public-health threat due to the emergence of praziquantel drug resistance.^[5,7,8] In addition, reported cases of reduced efficacy of praziquantel have been associated with multiple rounds of mass drug administration, highlighting the need for new antischistosomal drugs.^[9]

Compound libraries of pharmaceutical and academic organizations offer a potential source for identification of candidate therapeutics. However, the large number of drugs that need to be tested requires the use of medium- or high-throughput assays to implement an efficient and cost-effective screening.^[10,11] In addition to drug efficacy, information on pharmacodynamics would allow to select lead candidates. High activity, fast action, and low toxicity represent key parameters for the selection of promising leads during the in vitro screening process.^[10,12,13] Despite these requirements, the current gold standard for antischistosomal drug screening is based on worm phenotypic evaluation using operator-based microscopy, which is limited in throughput, labor-intensive, and subjective. Therefore, the development of advanced screening methods for the identification of new promising lead compounds is of high importance and priority.

In the last years, the more abundant Schistosoma mansoni larval-stage worms, or newly transformed schistosomula (NTS), have been used for the preselection of lead compounds prior to testing in adult parasites and to overcome the limitations related to the handling of adult worms for large-scale screenings.^[14,15] The advent of NTS-based screenings has set the stage for the development of automated and higher-throughput systems, and several screening methods have been explored. Microcalorimetry was found to be a useful tool to thoroughly analyze drug effects on adult and larval stage helminths in realtime, however it requires a large number of NTS per sample (at least 400).^[16,17] An alternate approach based on an image-based automated microscopy system was described as a label-free method to evaluate helminth viability based on morphology and motility.^[18,19] Nonetheless, it has not been adopted for systematic dose-response assays and real-time monitoring, as the high computational burden and the low level of parallelization limit its use for routine analysis. The impedance-based xCelligence system, which detects parasite viability in a 96-well plate format, has been applied to adult-stage S. mansoni but not to the highly abundant NTS.^[20] In summary, no drug screening system, which can automatically measure NTS viability and continuously assess dose-response effects, is currently available.

To address these current limitations, a first version of an in vitro impedance-based microfluidic assay for drug screening on NTS has been developed.^[21] Electrical impedance spectroscopy (EIS) is a noninvasive and label-free technique for investigating the dielectric properties of a sample and its potential variations within a specific frequency range.^[22,23] We previously demonstrated that the confinement of NTS in small sensing regions and the use of electrodes featuring dimensions comparable to those of the parasite larvae enable to assess NTS motility by means of EIS, and that this motility characterization may be a good indicator of NTS viability in drug screening applications.^[21] However, the rather complex microfluidic structures ultimately limited the analysis throughput, the measurements still required considerable sample handling by an operator, and the system did not allow for long-term culturing and real-time detection of drug-induced effects.

In this paper, we present a parallelized impedance-based platform to continuously assess dose-response effects of drugs on NTS. The electrical-impedance microwell (EIM) platform includes 32 analysis units to allow for simultaneous execution of several measurement replicates and to increase throughput. To achieve high sensitivity of the detection method without physical confinement of the parasites in channels of sub-millimeter dimensions, we used sensing electrodes with dimensions comparable to the NTS size, which resulted in a sensing volume of ≈25 nL or ≈100 μm height, given the well dimensions. This configuration enabled the use of a large medium reservoir over the sensing region to achieve long-term culturing of the parasites without affecting the detection characteristics of the system with respect to NTS motility. In addition, the chip design features simple loading of the parasites, which are driven into the nanoliter sensing volume by sedimentation. To validate our platform, we first measured the NTS response to three antischistosomal compounds, oxethazaine, praziquantel, and mefloquine, which have already been shown to cause different phenotypical behaviors in vitro, that is, they reduce parasite motility, stimulate hyperactivity or induce both effects, respectively.^[24–28] As a case study, we then analyzed the activity of an antipsychotic drug, methiothepine, which belongs to the emerging class of tricyclic compounds and serotonin modulators, which have been investigated as potential therapeutic agents against schistosomiasis.^[24,29] The obtained results indicate that the EIM platform can provide continuous dosedependent viability and activity patterns of NTS on-chip and can be a suitable component for antischistosomal drug screening.

2. Results

2.1. EIM Platform Function and Operation

The overall concept of the EIM platform is illustrated in Figure 1. To provide continuous impedance-based recordings of parasite motility in parallel to optical inspection, the analysis unit was fabricated from two transparent components: a PDMS (polydimethylsiloxane) microwell and a glass slide patterned with platinum electrodes. Each culture unit was loaded with 60 μL of NTS suspension (≈10–15 NTS per 60 μL) by using a pipette (Figure 1A-II). The NTS rapidly (≈1 min) settled to the bottom of the microwell (chamber) on the patterned glass substrate. Each microwell was equipped with a pair of coplanar electrodes for detecting NTS motility by measuring conductivity variations that were caused by parasite movements between the electrodes. To measure the signal fluctuations upon NTS activity and movements, an AC voltage was applied to the left co-planar electrode, and the current flowing through the sensing volume was then measured at the right electrode and converted to voltage through a trans-impedance amplifier (Figure 1A-III). Motile and non-motile parasites were evaluated separately in the device to detect differences in the voltage signal during 1-min acquisition. Because of the parasite movements, the high-pass-filtered signal output of motile NTS exhibits clear fluctuations around zero (Figure 1A-IV), while non-motile NTS do not cause signal variations, and the trace only shows the readout background noise (in the μV range, Figure S1, Supporting Information).

Figure 1. Detection of NTS parasite motility by the EIM platform.

A) The analysis unit of the EIM platform consists of an inverted-pyramid-shape PDMS part, aligned with a pair of platinum electrodes I). After loading and sedimentation of the schistosomula in the inverted-pyramid-shape PDMS well II), the impedance-based motility measurement of NTS was carried out III). Signal fluctuations, caused by impedance variations between the electrodes owing to larvae movements, were acquired using a 500 kHz sinusoidal excitation. The graph shows an example of 60 s of signal recordings for both, motile and non-motile parasites IV). The signal was filtered using a 0.2 Hz high-pass filter to remove the signal baseline. B) The power of the signals acquired from motile and non-motile NTS parasites was calculated in a bandwidth of 1–3 Hz during a time window of 1 min and measured every 15 min. The dotted line indicates the mean value of the signal power for motile (blue) and non-motile (red) NTS. The shaded areas show ±3 standard deviations. The power unit refers to 10⁻⁶ a.u. C) Bright-field microscopy images of motile and non-motile NTS parasites in the analysis units. The yellow arrows indicate parasites that contract and elongate between frames.

To assign a motility value as a function of signal fluctuations in a time window of 60 s, we computed the signal power in a bandwidth of 1–3 Hz. Figure 1B shows an example of the separation between the normalized signal power generated by motile and non-motile NTS, evaluated every 15 min during 1 h. The signal power measured with alive and motile parasites (−13.1 ± 1.1 dBμ, ≈0.05 a.u.) was, therefore, constantly two orders of magnitude higher than the signal power obtained from the non-motile counterpart (−34.7 ± 0.6 dBμ, in the range of 0.0005 a.u.), which evidences the robust recognition and differentiation of worm motility by using our platform.

The motility of the parasites was also qualitatively evaluated by visual inspection under an inverted microscope, as the chip was optically transparent and openings were included in the chip holder. The sequence of bright-field images clearly shows the contraction/extension movements of the alive NTS in between the electrode pair, while non-motile NTS do not move or change position (Figure 1C). The microscopy observations correlated with the impedance-based recordings and confirmed that the impedance signal fluctuations arose from the parasite movements.

2.2. Higher-Throughput EIM Platform Design

The higher-throughput EIM platform was designed to achieve parallelized and automated monitoring of the motility of NTS exposed to different test conditions. The platform included three components (Figure 2A): 1) A custom-made printed circuit board (PCB) for signal routing; 2) four analysis chips; 3) a device holder to accommodate the chips and provide connections between the PCB and the electrode pads on the chips. All these parts were designed according to a 96-well-standard format to ensure compatibility with standard lab equipment and automated imaging and analysis tools. The PCB enabled the multiplexing of the analog input/output signals of the impedance spectroscopy unit to any of the 32 parasite chambers. The PCB featured two analog multiplexers to separately switch the 32 input and output electrodes, spring-contact connectors for interfacing with the fluidic chips, a mini-HDMI plug to connect to the controller board outside of the incubator, and two analog SMA connectors to the input/output ports of the impedance spectroscope. The chips were inserted in a 3D-printed device holder with four insertion sites. Each chip included a top PDMS layer, containing the microfluidic structures that defined the 8 analysis units (microwell) and a bottom glass slide patterned with 16 co-planar platinum electrodes. To prevent absorption of small hydrophobic molecules from the solution into the PDMS during the drug assays, all chips were coated with a parylene-C layer (Figures S2 and S3, Supporting Information).^[30] Moreover, the microwells were arranged at 9 mm pitch to allow for loading of the parasites and of the medium by means of a standard multichannel pipette to simplify platform operation. Each analysis unit was shaped as an inverted pyramid to bring the parasites down to the micro-sensing area (450 × 450 μm²) through sedimentation and to provide enough medium volume (60 μL) for multi-day experiments. Despite the considerable liquid volume over the sensing area, the electrode dimensions limited the sensing volume to the first 100 μm in height, which corresponds to the approximate size of the NTS (≈50 × 100 μm²) (Figure S4, Supporting Information). The overall sensing volume (≈25 nL) was defined to enable testing with ≈10–15 NTS per condition, which represents an ≈tenfold reduction in sample consumption with respect to standard visual evaluation methods.^[12]

Figure 2. Design of the EIM platform.

A) The EIM platform can accommodate up to four chip units, which were placed on a custom-made PCB. A single chip (highlighted with dashed red line) hosted eight PDMS analysis units that were plasma-bonded to a glass substrate featuring platinum electrodes. The PDMS cavity featured an inverted-pyramid shape to promote sedimentation of the parasites to the sensing volume over the coplanar electrodes. B) The electrical equivalent circuit of the experimental setup, which included a lock-in amplifier for generating the AC stimulation signal (V_in), a custom-made PCB for routing the AC signal to the selected analysis unit, and a trans-impedance amplifier (TIA) for current-to-voltage conversion. The voltage signal was sampled by the lockin amplifier and recorded. A controller board featuring a microcontroller (MCU) was used for synchronizing the switching between the analysis units and the lock-in amplifier.

The automated multiplexing in the platform allowed to simultaneously record from up to 32 analysis units. From an electrical point of view, each sample can be described as a variable impedance Z_s, representing the NTS suspension between the electrodes, in series with two double-layer capacitances, C_dl, which form at the interface between the electrodes and the medium (Figure 2B). The variations of Z_s over time were caused by the movement of the NTS in between the electrodes, while the Z_s average value depended on the solution conductivity and on the number of parasites between the electrodes. A multiplexing and switching architecture was used to route the output of the lock-in amplifier to each of the 32 parasite chamber units. Correspondingly, a de-multiplexing stage was used to route the output signals of each chamber to the input of the trans-impedance amplifier (TIA). The frequency of the sinusoidal carrier signal (500 kHz) was selected to enable fast multiplexing and to minimize the signal attenuation in the EIM platform (≈−32 dBV, Figure S5, Supporting Information). The switching interval and the selection of the electrode pairs were defined by a custom-made Python script, which interfaced to a microcontroller (MCU, on the controller board) that controlled the de/multiplexers on the PCB. To achieve continuous and quasi-parallel impedance measurements of the NTS in the 32 chambers, we performed short recordings (1 ms) at each electrode pair and fast switching (1.5 μs) between all the units in a round-robin fashion, which resulted in an effective sampling frequency of ≈32 Hz per chamber unit.

2.3. Monitoring of Parasite Motility Under Drug Treatment

To validate the ability of the EIM platform to detect NTS motility and to discriminate between different schistosomula movement characteristics, we measured the signal fluctuations caused by the parasites following incubation with two compounds, known to affect schistosomula phenotypes. We selected oxethazaine, a fast-acting in vitro compound that reduces parasite motility,^[24] and praziquantel, which, although mostly effective against adult parasites, is known to have an excitatory effect on NTS in vitro.^[26] The parasites were exposed to different concentrations of these test compounds, and we tested our impedance-based motility readout. As controls, we measured also the motility of NTS under standard medium conditions and in medium containing the drug vehicle (DMSO). The parasite larvae were first loaded in the chips under standard medium condition using a multichannel pipette. We then performed impedance measurements of all the chambers during 1 h to evaluate the motility of the untreated parasites and to confirm their initial viability (Figure 3A). After baseline acquisition, the different drug concentrations were added to the chamber units and NTS motility was recorded over time. This procedure enabled us to normalize the power of the signal fluctuations to the power of the first 1-h window for each condition and to extract a motility index. This normalization also allowed for comparing measurements from chambers with different numbers of parasites, as the absolute signal power depends on the number of loaded parasites.

Figure 3. Measurements of drug-induced NTS parasite motility via impedance-based recording.

A) The flow diagram shows the main steps for performing an impedance-based drug assay. First, 30 μL of NTS solutions were loaded into all analysis-unit chambers. After 1 h of impedance-based motility detection, the measurement was stopped and additional 30 μL of drug solution were dispensed into each chamber. Afterward, the impedance detection was restarted and recordings of drug-induced motility variations continued until the end of the assay. B) The graphs show the variations in the motility index of the vehicle-control sample (exposed to 0.5% v/v DMSO) and of NTS exposed to four different concentrations of oxenthazaine (left) and praziquantel (right). For clarity, one point per hour is displayed, and each point indicates the mean value of the motility index determined from three analysis units. Five trend lines were superimposed to the points to guide the eye for the different tested conditions. C) The motility graphs of the first 2 h of recording for both, oxenthazaine and praziquantel. Each point represents the mean value of the motility index simultaneously measured with three analysis units, while the error bars represent the standard error of the mean.

Our platform allowed to differentiate the inhibitory and excitatory effects, induced by the two test compounds (Figure 3B). After addition of oxethazaine (t = 0 h) at all concentrations tested, the NTS exhibited a considerable decrease in motility, compared to the vehicle control condition, during 48 h of continuous measurements. The reduction of NTS motility showed a drug-dose dependence: the motility index for 6 μm oxethazaine reached zero (i.e., no motility) in 12 h, whereas the 3 μm condition required about 24 h to achieve the same effect. The highest drug concentration, 12 μm, inhibited the larvae movement within the first 15 min upon addition of the compound. Contrarily, praziquantel showed a drastic increase in NTS motility, which is in line with the phenotypical evaluations of drug effects reported in literature.^[25,26] Higher motility index values (≈1.5) were obtained for 1.5 and 3 μm of praziquantel. After reaching a peak of the motility index, all the drug–dose curves exhibited a gradual decrease in motility and approached the vehicle-control behavior in about 18 h. Moreover, the effect of both drugs was also confirmed in a 96 well-plate by the standard visual method (Movies S1 and S2, Supporting Information).

By focusing on the first 2 h of the motility detection for both compounds, we evaluated the sensitivity of the platform in discriminating motility changes caused by different drug concentrations (Figure 3C). To observe drug-induced rapid variations of NTS behavior, the motility index was computed for 1-min windows every 15 min during the entire recording. We achieved a robust discrimination of the effects induced by 3, 6, and 12 μm of oxethazaine after 75 min compared to the 0.5% v/v DMSO and 1.5 μm oxethazaine conditions. For praziquantel, we observed a significant increase in motility after 30 min for all tested concentrations in comparison to the vehicle-control condition.

2.4. Long-Term Dose-Response Analysis

To evaluate the performance of our platform in continuously assessing NTS viability and generating real-time dose–response curves, we exposed the parasite larvae for 48 h to six serially diluted concentrations (1.5, 3, 6, 12, 25, 50 μm) of two compounds, oxethazaine and mefloquine, which are known for their antischistosomal activity in vitro.^[24,28] The impedancebased parasite viability was calculated by normalizing the motility index of each drug condition to the motility index of the vehicle control at each time point along two days of drug exposure.

By measuring parasite viability exposed to oxethazaine via the EIM platform, no signal fluctuations were detected for the NTS incubated with the highest drug concentrations (12, 25, and 50 μm) after 30 min, indicating that the NTS were dead (Figure 4A). For the same time point, the viability upon dosage of 6 μm was already reduced to ≈0.5, which indicates that a significant fraction of the NTS population has deceased or lost its motility. After 17 h, only the parasites exposed to 1.5 μm showed a viability above 0.5 (0.54 ± 0.05), which further decreased to ≈0.2 (0.2 ± 0.19) at 48 h of incubation with the drug. By testing the activity of mefloquine on the NTS, we obtained dose–response curves along time that evidenced a lower potency compared to oxethazaine. After 3 h, only the parasites exposed to 50 μm were dead, whereas those exposed to 25 μm showed a viability slightly below 0.5 (0.39 ± 0.21). Toward the end of the drug assay (39 h), only the parasites exposed to 6 μm displayed a viability of 0.37 ± 0.19, which further decreased to 0.15 ± 0.08 at 48 h. Parasites exposed to drug concentrations lower than 6 μm were viable and showed a high level of motility during the entire assay.

Figure 4. Temporal evolution of the dose–response curves determined through continuous long-term monitoring of NTS viability by impedance-based detection.

A) The graphs show the impedance-based estimations of the NTS viability at four selected time points as a function of drug concentration. The NTS was exposed to six different concentrations (1.5, 3, 6, 12, 25, and 50 μm) of oxethazaine (left) and mefloquine (right) during 48 h. Each circle represents the mean value of the viabilities of three replicates, and the error bars represent the standard error of the mean. The four sigmoid fits, used to calculate the IC₅₀, values are superimposed to the viability measurements. B) The temporal evolution of the IC₅₀ values for oxethazaine and mefloquine as determined by using impedance-based detection. The IC₅₀ values obtained through standard visual scoring at 24 and 48 h are shown for comparison (marked in red). The circles represent the values obtained through the sigmoidal fits, while the error bars show the 95% confidence intervals.

We used the viability calculations to extract IC₅₀ values of oxethazaine and mefloquine (Figure 4B). IC₅₀ corresponds to the drug concentration at which the larvae were compromised in their activity and moving 50% less. Sigmoid fits (shown in Figure 4A) were used to calculate the IC₅₀ values over time. The IC₅₀ values obtained from the impedance-based viability measurements showed that oxethazaine featured an IC₅₀ of 6.1 ± 0.76 μm at 30 min incubation. The IC₅₀ value rapidly decreased and reached 2.02 ± 0.16 μm after 12 h of drug exposure. Between 24 and 48 h, the IC₅₀ did not change significantly anymore (1.51 ± 0.21 μm and 1.45 ± 0.34 μm, respectively). In the case of mefloquine, the impedance-based estimations evidenced an IC₅₀ of 24 ± 1.42 μm after 3 h of exposure, which decreased linearly to 8.51 ± 1.07 μm during 24 h of incubation. Finally, the IC₅₀ calculated at 48 h using the EIM platform was 5.05 ± 0.21 μm.

The parasite viability was also evaluated using the standard visual scoring method at 24 and 48 h in a 96-well plate to validate the IC₅₀ estimations obtained with the EIM platform (Movies S1–S3, Supporting Information). The IC₅₀ concentrations of oxethazaine obtained from the visual evaluation were 1.39 ± 0.06 μm after 24 h and 1.23 ± 0.04 μm after 48 h. In the case of mefloquine, the IC₅₀ estimations from the visual method were 2.28 ± 0.22 μm at 24 h, and 2.03 ± 0.16 μm at 48 h. The visual-score-based evaluations were in good accordance and within the same order of magnitude as the results obtained with the impedance-based method (Figure S6, Supporting Information).

2.5. Characterization of Methiothepine Effect on NTS

As a case study, we challenged our system by investigating the effect of methiothepine on schistosomula. Methiothepine is a tricyclic antipsychotic compound whose behavior on NTS has not been fully characterized in vitro.^[24] Tricyclic antipsychotic compounds bind to the serotonin transporter (SERT) of helminths and have been studied for their antischistosomal effects.^[31] It has been previously shown that SERT antagonists can cause either persistent or transient hyperactivity of helminths and, eventually, can affect parasite viability.^[32] To explore the temporal evolution of the dose-response effect, we measured the motility index and the viability of NTS exposed to six different methiothepine concentrations (1.5, 3, 6, 12, 25, 50 μm) for 48 h. We observed an excitatory effect lasting ≈24 h that was induced by the drug at sub-lethal concentrations (<25 μm at 24 h; Figure 5A), which is in line with previous observations of NTS exposed to other members of the tryclic-compound family.^[31,32] Live imaging of parasites in the chamber unit confirmed the hyperactivity of NTS that were incubated with sublethal methiothepine concentrations (Figure 5B).

Figure 5. Characterization of NTS parasite motility and viability during long-term exposure to methiothepine.

A) The motility values of the NTS, exposed to four different concentrations of methiothepine, are shown and compared to the motility indices of the vehicle-control sample (NTS exposed to 0.5% v/v DMSO). Each point represents the mean value of the motility indices of three replicates. Trend lines were superimposed to the points to guide the eye for the different tested conditions. B) Time-lapse images of NTS showed qualitative movement differences between NTS exposed to 6 μm methiothepine and to 0.5% v/v DMSO. The yellow arrows indicate parasites that moved between the different frames. C) The impedance-based viability measurements of NTS at four selected time points are plotted as a function of the drug concentration. The NTS was incubated with six different concentrations of methiothepine (1.5, 3, 6, 12, 25, and 50 μm) during 48 h. Each circle represents the mean viability value of three replicates, and the error bars represent the standard error of the mean. The sigmoid fit was used to calculate the IC₅₀ value at each time point. D) IC₅₀ values, calculated from the impedance-based detection of NTS viability, are compared with IC₅₀ values obtained by standard visual scoring at 24 and 48 h (marked in red). The circles represent the values determined from the sigmoid fits, while the error bars show the 95% confidence intervals.

After 4 h of incubation, only the 50 μm-condition caused a drastic decrease in NTS viability (0.2 ± 0.09; Figure 5C). A comparably high level of NTS viability was maintained over 30 h for lower doses, while, for longer exposure times, methiothepine showed a slow-acting and dose-dependent killing behavior. At the end of the assay, after 48 h of incubation with the drug, all concentrations higher than 1.5 μm exhibited a viability below 0.5. The extraction of the IC₅₀ over time also showed the slow efficacy of methiothepine in killing the NTS (Figure 5D). To inhibit the parasite viability by 50% during the first 24 h, a concentration of more than 26.81 ± 0.94 μm of methiothepine was required. The IC₅₀ calculated after 48 h using the EIM platform was 2.45 ± 0.9 μm. The IC₅₀ values extracted by the standard visual evaluation amounted to 10.88 ± 2.05 μm at 24 h and 2.22 ± 0.31 μm at 48 h. Differences in IC₅₀ values obtained at 24 h by the two methods were expected, as the hyperactivity of the NTS introduces additional difficulties to visually score the parasite status for the operator (Movie S4, Supporting Information). Nevertheless, the IC₅₀ values obtained at 48 h with the two different methods are in good accordance.

3. Discussion

Advancing the development of automated and medium- or high-throughput approaches for antischistosomal drug screening is of fundamental importance for the identification of new compound candidates. In this work, we introduced a novel platform with integrated electrodes for the automated detection of schistosomula viability by means of an impedance-based method. In particular, we showed how this label-free technique offers an unbiased method to quantitatively score parasite viability, and that the impedance method enables long-term and continuous assessment of drug efficacy.

The EIM platform allows for robust and simple screening of the viability of NTS that have been exposed to different drug concentrations, and it requires minimal operator interference. The use of micron-size electrodes and the small detection volumes reduces the number of NTS that are needed for analysis ≈tenfold in comparison to the current standard visual evaluation method,^[12] and up to 30-fold in comparison to luminescence- and fluorescence-based assays reported in literature.^[33,34] The arrangement of the pyramid-shape wells facilitates the loading of the parasite samples with standard multichannel pipettes and ensures the positioning of the parasites on top of the electrodes for over 48 h. In addition, the 96-well format of the platform enables compatibility with standard lab automation tools, such as automated liquid handlers, which further improves automation and increases the throughput.

The impedance-based readout allows to overcome major limitations associated with the currently used visual evaluation method, which include limited throughput, potential subjectivity, and bias in the operator's scoring.^[14,35] In a previous proof-of-principle study, we showed the possibility to use impedance measurements of NTS motility as proxy for parasite viability.^[21] However, the previous system did not allow for long-term culturing of NTS and could only provide end-point evaluation of the larvae viability. Furthermore, the complex fluidic structure that was used to confine the larvae in the sensing area and to increase the signal-to-noise ratio of the measurements strongly limited the analysis throughput to four recordings in parallel. Here, we were able to preserve the previously demonstrated high sensitivity of the impedance-based characterization by confining the larvae to a small detection volume in an easy-to-operate open fluidic structure. This solution enabled to simplify the platform operation and increase the analysis throughput to up to 32 recordings in parallel. We also determined the Z′-factor, which indicates the quality of an assay based on the difference of mean values of positive and negative controls in reference to the corresponding standard deviations.^[36,37] The developed system showed a Z′-factor of 0.63 (calculated from 32 × 4 measurements of pre-treated motile and dead parasites of the impedance-based assays, power_motile = −12.8 ± 1.9 dBμ and power_dead = −34.5 ± 0.8 dBμ), which highlights the potential of the platform as a robust and higher throughput antischistosomal screening method according to NIH guidelines.^[36] The novel design also enabled the long-term culturing of the parasite larvae to realize a real-time evaluation of drug activity, which is important for providing insights into drug kinetics and for selecting fast-acting compounds.^[38] High and fast drug activity along with low toxicity are relevant criteria to select promising antischistosomal lead compounds.^[24]

The constant decrease of motility in the control samples during long-term in vitro culture, a common behavior of NTS also observed in standard assays, may introduce artifacts in the evaluation of compound efficacy in multi-day drug-exposure experiments using motility-based parasite evaluation. Therefore, medium exchanges may be considered to extend the viability time of the NTS in the platform for long-term assays.

We first validated the EIM platform by recording variations in NTS motility upon dosage of three active drugs triggering antischistosomal effects (Figure S7, Supporting Information). By analyzing the effect of praziquantel on NTS motility, we were able to reproduce the in vitro excitatory action of the drug on parasites, which lasted for over 18 hours. This hypermotility is caused by the activity of praziquantel as calcium channel agonist, which increases the calcium concentration in the schistosomula body within minutes of exposure and causes intense and sustained muscular contraction of the parasites.^[26,39] In contrast, the exposure of NTS to oxethazaine caused a fastacting inhibitory effect on their motility.^[24] Motility reduction is most likely caused by the high affinity of the drug to the sodium channels of the NTS, which has been shown to have an in vitro inhibitory effect on the schistosomula smooth muscles by blocking the action of serotonin.^[27] Via our impedance-based detection, we were able to confirm the fast action and the high efficacy of the drug in vitro, which showed an IC₅₀ value of 3.48 μm already after 1 h of incubation. To investigate complex antischistosomal drug response, we studied the effect of mefloquine on NTS with the EIM platform. This antimalarial compound is known to induce hyperactivity and to affect NTS viability in vitro, which may be attributed to its potential role as an inhibitor of glycolysis and interference with schistosomula metabolic activity.^[40] Our impedance-based IC₅₀ estimations confirmed the reported lethal effect of mefloquine on NTS showing values below 10 μm after 20 h of drug exposure.^[21] Differences in IC₅₀ values, obtained with impedance-based and visual methods were expected for mefloquine at 24 h, as the morphology of the NTS was highly affected at high drug concentrations, and it was difficult to detect subtle movements of the larvae by eye, which, however, remained detectable using the impedance system.^[21,28]

After validating the ability of our method in detecting changes of NTS motility caused by drugs with known in vitro effects, we used the EIM platform to analyze the response of the schistosomula to methiothepine. The complex dose- and time-dependent response of the NTS to the compound underlines the importance of continuous monitoring of parasites when investigating the efficacy of new drugs. Methiothepine is an antipsychotic drug of the tricyclic group, which acts as an inhibitor of both the serotonin receptor and the SERT, depending on the dose.^[24,29] A transient increase in schistosomula motility was detected for sub-lethal drug concentrations (<12 μm of methiothepine for the first 24 h of incubation). This result corroborates the effects observed for two classical SERT inhibitors, fluoxetine and clomipramine, which were shown to induce strongly hyperactive phenotypes.^[41] Recently, incubation of NTS with paroxetine, another S. mansoni SERT inhibitor, in the 1–10 μm concentration range was reported to have a similar hypermotility effect during the first 24 h of incubation, while the motility then decreased for longer exposure times.^[32] Our measurements demonstrate that methiothepine has a similar effect on NTS for an analogous concentration range, as the schistosomula showed decreased motility after 24 h. Higher concentrations of methiothepine (>12 μm) resulted in a rapid loss of motility and a consequent reduction in larvae viability,^[42] which confirmed earlier findings^[24] and was similar to what has been previously reported for high concentrations of paroxetine.^[32]

In summary, we developed a parallelized and automatable drug-screening platform, which continuously provides dose-dependent viability scores of NTS. The drug-dose responses of the parasites to four different antischistosomal compounds that were obtained through impedance detection show good agreement with those obtained from standard visual scoring of NTS motility. This agreement evidences that the impedance-based approach constitutes a reliable alternative method to identify novel drug candidates in vitro. The current platform layout enables the operator to use up to 4 chips, which include 32 analysis units in parallel, with a single instrument. The electrical detection method allows for further parallelization to achieve increased throughput with minimal experimental-setup modifications. The chip design, implemented in PDMS, can be easily realized with standard plastic materials to avoid issues related to compound ad/absorption in PDMS, and fabrication processes for mass production, such as injection molding, can be used. Finally, the EIM platform can be readily adapted to other relevant motile schistosome stages, such as cercariae, juvenile and adult parasites, or to different parasite species by simple modification of the sensing-area design, which will contribute to improve anthelmintic drug-screening applications.

4. Experimental Section

Culture Medium and Drugs

M199 medium was obtained from Gibco (cat. no. 22340-020, Thermo Fisher Scientific, Waltham, USA). Penicillin/streptomycin 10’000 U mL⁻¹ (pen/strep, cat. no. P4333-100ML, Sigma-Aldrich, Buchs, Switzerland) and inactivated fetal calf serum (iFCS, cat. no. 2-01F30-I, Bioconcept AG, Allschwil, Switzerland) were purchased from Bioconcept AG. All media were sterilized by filtration using a 0.22 μm filter bottle (cat. no. 431097-COR, Vitaris AG, Baar, Switzerland). Oxethazaine (cat. no. O5380-5G, Sigma-Aldrich, Buchs, Switzerland), praziquantel (cat. no. P4668-5G, Sigma-Aldrich, Buchs, Switzerland), mefloquine (cat. no. M2319-100MG, Sigma-Aldrich, Buchs, Switzerland), and methiothepine (cat. no. M149-100MG, Sigma-Aldrich, Buchs, Switzerland) were all purchased as racemic powders from Merck (Sigma-Aldrich).

Parasite Culture and Transformation

S. mansoni culturing and NTS transformation were performed according to protocols previously described in literature.^[12,43] In brief, S. mansoni -infected Biomphalaria glabrata snails were placed singularly in 24-well plates and exposed to a neon lamp (36 W, 4000 K, 3350 lumens), for 3–4 h, to induce the shedding of cercariae. The supernatant was collected and filtered to remove impurities in the solution. The mechanical transformation of the cercariae into NTS was performed by physically removing the tail by constricted passage through a Luer-Lok tip in between two 12 mL syringes. The NTS was resuspended in M199 medium supplemented with 5% v/v iFCS, 1% pen/strep, and 1% antifungal mix.

In Vitro Antischistosomal Drug Assay

The drug assay in the EIM platform was performed by first dispensing 30 μL of NTS solution with 1 NTS 2 μL⁻¹. After 1 h recording of the NTS-induced fluctuations as baseline activity, 30 μL of drug solution (1.5–50 μm in NTS medium) were added, yielding a total volume of 60 μL in each analysis unit. Final drug concentrations ranged between 0.75 and 25 μm in a 2× dilution series. In each assay, 0.5% v/v DMSO (vehicle control) and blank M199 medium controls were included. All conditions were measured in quadruplicates. NTS viability under each test condition was measured every 15 min for 48 h in the EIM platform. A viability score ranging from 0 to 1 (0 = non motile and dead parasite, 1 = motile and alive parasite) was assigned to each test condition and per time point, according to the relative motility of the parasites with respect to the vehicle control motility (see Data Analysis for more information).

In parallel, identical drug concentrations were set up in a 96-well plate (cat. no. 83.3924, Sarstedt, Nümbrecht, Germany) to perform the same experiment by using the standard visual scoring method to compare the results obtained via standard viability detection with the impedance-based characterization. 50 μL of 2 NTS 1 μL⁻¹ suspension were dispensed in each well, and subsequently 50 μL of drug-concentration solutions were added to reach a final drug concentration ranging from 0.75 to 25 μm. Each condition was prepared in duplicates or triplicates. A trained operator evaluated the viability of the NTS in the different drug solutions assigning a score from 0 to 3 on a quarter-of-a-point scale. In this scale, 0 represents NTS with complete loss of motility, while 3 represents NTS with good motility, good overall viability, and healthy behavior.^[12] Visual scoring was carried out after 24 and 48 h of drug incubation.

Each drug test was performed in a separate experiment.

Chip Fabrication

The analysis chip consisted of two parts: a polydimethylsiloxane (PDMS) layer containing the microfluidic structures and a glass slide with a patterned metal layer. The PDMS layer was cast from a 3D-printed master mold (fabricated in Accura SL 5530, Protolabs, Feldkirchen, Germany) by using soft lithography. The silicone and curing agent (Sylgard 184, Dow Corning Corp., Midland, USA) were mixed at a 10:1 w/w ratio, degassed, and poured onto the master mold. After curing or 2 h at 85 °C, the PDMS layer was peeled off the master mold and cut into individual chips.

The 200-nm-thick platinum electrodes were deposited on a 6-in., 500-μm-thick borosilicate glass wafer via a lift-off process. Briefly, the wafer was spin-coated with lift-off resist (LOR3B, Microchem Corp., Newton, USA), followed by a positive photoresist (S1813, Rohm-Haas, Schwalbach, Germany), and patterned using photolithography. After Pt deposition, the lift-off of the metal was carried out by using Mr-Rem-400 remover (micro resist technology GmbH, Berlin, Germany). Finally, the glass wafer was diced into individual glass slides (20 mm × 62 mm).

Each PDMS chip and patterned glass slide were aligned using a custom-made alignment tool and irreversibly bonded together after surface treatment using oxygen plasma (Harrick Plasma PDC-002, Harrick Plasma, Ithaca, USA).

Parylene Coating of PDMS Chip

To prevent drug absorption by PDMS during long-term compound incubation (48 h), we coated the chip surface with parylene-C polymer.^[30,44] All microfluidic chips were coated using a parylene coating system (Parylene P6, Diener Electronic GmbH, Ebhausen, Germany). The devices were placed in the center of the rotating trays in the deposition chamber. 10 g of parylene-C dimer powder (parylene, Diener Electronic GmbH, Ebhausen, Germany) were placed into the evaporator. The system was evacuated to 0.012 mbar before the deposition was initiated. The powder was evaporated in a temperature range of up to 170 °C, cleaved in the pyrolysis tube at 720 °C, and deposited onto the samples at 80 °C and 0.03 mbar over the course of 4.2 h (Figure S2A, Supporting Information). After the evaporation of the parylene powder, the deposition chamber was cooled down to 41 °C and the devices were then removed from the coating system.

The thickness of the deposited parylene-C layer was evaluated by using a 3D optical surface profiler (Zygo Corporation, Middlefield, USA) on a microscope slide (Menzel-Gläser, Thermo Scientific, Dreieich, Germany), placed in the chamber with the devices (Figure S2B, Supporting Information). For this purpose, the parylene layer was cut, one part was pealed from the glass slide, and the thickness was measured along the cut.

Rhodamine Absorption Evaluation

To compare the dye absorption in parylene-coated PDMS and bare PDMS chips, 30 μL of rhodamine B (83689, Sigma-Aldrich, Buchs, Switzerland) solution (0.1 mm in DI water) were loaded into each well chamber. Fluorescence images inside the chambers were captured before the loading of the dye, at 5 min, 1 and 2 h after sample loading using an inverted microscope (Nikon Ti-E, Nikon, Egg, Switzerland). During the measurements, the chips were kept in the dark to prevent photobleaching and at 37 °C, 5% CO₂ using a stage-top incubator. The microscope was controlled using Youscope software, and offline image analysis was performed using ImageJ (Figure S3, Supporting Information).

EIM Platform Assembly

The chips were placed between a custom-made printed-circuit board (PCB) and a chip holder (Figure S8, Supporting Information). The PCB was designed in Altium Designer 17.0 and ordered from PCBWay (Hangzhou, China). Electrical connections between the PCB and the analysis chips were obtained by contacting the electrode pads from above using spring-loaded pins (0956-0-15-20-75-14-11-0, Mill-Max Mfg. Corp., Oyster Bay, USA). The PCB featured four window-like openings (15 mm × 58 mm) to allow for visual access to the chips without disassembling the platform.

The chip holder was 3D-printed by means of stereolithography (Protolabs, Feldkirchen, Germany) in ABS-like material (Accura Xtreme White 200, Protolabs, Feldkirchen, Germany). The printing material was selected as to withstand the high-humidity conditions in the incubator and the high force levels required for reliable chip connection. The chip holder also featured four openings to allow for visual examination of the parasites using a standard inverted microscope.

Neodymium block magnets (Q-10-03-02-HN, Supermagnete, Uster, Switzerland) were used to align and keep the chip holder and PCB in position, and to provide the force necessary for pressing the spring-loaded pins onto the electrode pads on the chips for stable electrical connection. The magnets were attached to the chip holder and the PCB with a 2-component white epoxy adhesive (EA 9492, Henkel, Düsseldorf, Germany). Finally, the platform was covered with an omniTray lid (Nunc OmniTray Single-Well Plate, Thermo Fisher Scientific, Reinach, Switzerland) to prevent medium evaporation from the culture units.

Impedance Measurement

Impedance measurements were performed using a HF2-LI impedance spectroscope (Zurich Instruments AG, Zurich, Switzerland). The analysis chips were contacted via a custom-made PCB to route the connections from the impedance spectroscope to the integrated electrodes. An AC voltage with an amplitude of 100 mV and a frequency of 500 kHz was applied between the selected pair of coplanar electrodes. The current flowing through the system was then converted to voltage through a trans-impedance amplifier (HF2TA, Zurich Instruments AG, Zurich, Switzerland) with a 1-kΩ feedback resistor and sampled by the HF2-LI with a sampling frequency of 14 kHz. The acquired signal was filtered with a 2.2-kHz low-pass filter in the impedance spectroscope. The amplitude variation of the sampled current-to-voltage signal was then used for further analysis. A custom-made software, written in Python, was used to control the selection of the electrode pair and to control the signal acquisition.

Microscopy

During the recording of the impedance signals, the platform was placed on the stage of an automated microscope. Microscopic images were obtained using an inverted microscope (Nikon Ti-E, Nikon, Egg, Switzerland), placed in an environmental control box, which maintained a stable temperature of 37 °C, CO₂ of 5%, and a relative humidity of ≈90%. Bright-field images were captured on the Nikon microscope using a Nikon Plan Fluor 10X objective (NA 0.3, WD 16 mm). Automated imaging was performed for 20 s every hour from each analysis unit during the entire experiment. The live micrographs were recorded to compare the results of the gold-standard evaluation (visual scoring) with those obtained by the impedance-based readout. The microscope was controlled using Youscope software, and offline image analysis was performed using ImageJ.

For standard visual assessment, the parasites were incubated in culture medium with the test compounds in a 96-well plate in duplicates/triplicates for 48 h. Every 24 h, the drug effects on the parasites were assessed by visual scoring with a light microscope using a magnification of 4–10×.

Computational Modeling

A finite-element-method (FEM) model was used to verify the current-density distribution in the microwell (Figure S4A, Supporting Information). The current passing through the electrodes in the measurement chamber was calculated by integrating the current density over the middle orthogonal cross section area of the microwell in Comsol Multiphysics 5.4 (COMSOL AB, Stockholm, Sweden). To evaluate the current density across the entire depth of the microwell, the current was derived over the microwell height (Figure S4B, Supporting Information).

Data Analysis

The voltage-converted current signals were processed and analyzed in MATLAB (The MathWorks Inc., Natick, USA). The recorded signals were filtered using a 0.2-Hz high-pass filter to remove slow signal variation due to solution evaporation. Each analysis unit may exhibit different baseline values due to differences in solution conductivity caused, for example, by different drug compounds or drug concentrations, or by subtle variations in the alignment of the electrodes with the microwell base. To reduce the influence of such effects, the high-pass-filtered traces were normalized with respect to the mean baseline signal of the respective unit. To quantify the signal fluctuations induced by the parasites, the power of the filtered and normalized signal was computed in a 1–3 Hz bandwidth. This approach minimizes the effect of readout noise, which is present at higher frequencies, while it preserves the signal power that is related to the movement of the NTS between the electrodes. To confirm that the sample was correctly loaded in the microwell, the signal power was first measured during 1 h prior to addition of the drug compound. Only analysis units, the signal power of which ranged between (−18.5, −7.1) dBμ (± 3 standard deviations of the average NTS motile value, −12.8 ± 1.9 dBμ), were further analyzed. The runtime calculated power of the measured fluctuations in each unit was normalized to its initial power magnitude (t_-1hr) in order to compare measurements with different numbers of NTS in the sensing compartments and to extract motility index parameters. To evaluate NTS viability, the motility index of every condition was normalized to the motility index of the vehicle control. This procedure followed the normalization performed in the standard drug assay using visual scoring and it helped to remove NTS phenotype variations caused by the vehicle.^[21] In addition, a viability value of 1 was assigned to all the microwells in which the NTS showed a motility equal or higher than that in the vehicle control microwells.

The viability scores obtained from visual evaluation were averaged across replicates and normalized to the vehicle-control viability score.

The half-maximum inhibitory concentration (IC₅₀) values of the tested drugs were determined for both visual-inspection-based and impedance-based viability scores by applying a nonlinear least-squares analysis. A two-parameter sigmoid function with a constant hill slope was fitted to the viability scoring data. A single average slope was first computed across all experimental time points for each drug and then applied to the fit for the estimation of the IC₅₀ during the continuous long-term measurements.

The Z′-factor of the impedance-based assays was computed to determine the suitability of the EIM platform for high-throughput screening applications.^[36,37] The Z′-factor is defined as

$${\text{Z}}^{\prime }-\text{factor}=1-\frac{3{\sigma }_{+}+3{\sigma }_{-}}{\left|{\mu }_{+}-{\mu }_{-}\right|}$$

where μ+ and μ− indicate the mean signal power of alive (motile and pre-treated) and dead (non-motile) schistosomula and σ+ and σ− the corresponding standard deviations.

Supplementary Material

Supporting Information is available from the Wiley Online Library or from the author.