Planar Patch Clamp and Techniques of Patch-Clamp Spectroscopy in Antimicrobial Therapy

Introduction

Planar patch-clamp is an effective screening tool in pharmacology, institutionalized in molecular microbiology over 15 years ago [1]. The creation of planar polymer patch-clamp electrodes and silicon substrates in the 2000s [2,3] and the creation of microfluidic chips and perfusable chambers corresponding to this pharmacological screening technology, although works on the development of microfluidic chips for planar patch-clamp were already being published in the early to mid-2000s, and the first "benchmark study" of a chip for planar patch-clamp dates back to 2003 [4,5]. At the moment, there are automated and robotic schemes for planar patch-clamp, including those available for sale and maintenance [6-8]. In the CIS, this method is not widespread, however, devices such as the "Patchliner" with planar patch-clamp, capable of recording up to 8 cells synchronously, are available [9-11]. Patch-clamp tools can be used in the development of drugs and antimicrobial agents, screening of which can be performed on E. coli, etc. [12-14]. The response of the organism mediated by lysosomes can also be the subject of using this method, as lysosomal ion currents can also be measured in planar patch-clamp systems [PMID: 21139138].

Materials and Methods

In our work in 2017 ("Cellular Therapy and Transplantation"), a DIY planar patch-clamp system created in Russia based on components imported from Germany was tested. In this work, open-access planar patch-clamp recording files are used. Special DSP methods, different from Fourier transformation, are proposed/validated as methods of analysis and diagnosis on neural networks.

Results

It is shown that "patch-clamp spectroscopy" (obtaining spectra of patch-clamp data using DSP methods) with multiple cells is a representative approach applicable in cellular diagnostics. It is also possible to perform an equivalent analysis of reactive cytophysiological functions of lysosomes in antimicrobial therapy (using a chip model of planar patch-clamp) and the functionality of bacterial protoplasts.

References

1. Brüggemann A, Farre C, Haarmann C, Haythornthwaite A, Kreir M, Stoelzle S, et al. Planar patch clamp: advances in electrophysiology. Methods Mol Biol. 2008; 491: 165-76.
2. Klemic KG, Klemic JF, Sigworth FJ. An air-molding technique for fabricating PDMS planar patch-clamp electrodes. Pflugers Arch. 2005; 449: 564-72.
3. Nagarah JM, Paek E, Luo Y, Wang P, Hwang GS, Heath JR. Batch fabrication of high-performance planar patch-clamp devices in quartz. Adv Mater. 2010; 22: 4622-7.
4. Xu J, Guia A, Rothwarf D, Huang M, Sithiphong K, Ouang J, et al. A benchmark study with sealchip planar patch-clamp technology. Assay Drug Dev Technol. 2003; 1: 675-84.
5. Li X, Klemic KG, Reed MA, Sigworth FJ. Microfluidic system for planar patch clamp electrode arrays. Nano Lett. 2006; 6: 815-9.
6. Milligan CJ, Li J, Sukumar P, Majeed Y, Dallas ML, English A, et al. Robotic multiwell planar patch-clamp for native and primary mammalian cells. Nat Protoc. 2009; 4: 244-55.
7. Milligan CJ, Möller C. Automated planar patch-clamp. Methods Mol Biol. 2013; 998: 171-87.
8. Obergrussberger A, Brüggemann A, Goetze TA, Rapedius M, Haarmann C, Rinke I, et al. Automated patch clamp meets high-throughput screening: 384 cells recorded in parallel on a planar patch clamp module. J Lab Autom. 2016; 21: 779-93.
9. Farre C, Haythornthwaite A, Haarmann C, Stoelzle S, Kreir M, George M, et al. Port-a-patch and patchliner: high fidelity electrophysiology for secondary screening and safety pharmacology. Comb Chem High Throughput Screen. 2009; 12: 24-37.
10. Polonchuk L. Toward a new gold standard for early safety: automated temperature-controlled hERG test on the PatchLiner. Front Pharmacol. 2012; 3: 3.
11. Becker N, Horváth A, De Boer T, Fabbri A, Grad C, Fertig N, et al. Automated dynamic clamp for simulation of IK1 in human induced pluripotent stem cell-derived cardiomyocytes in real time using patchliner Dynamite8. Curr Protoc Pharmacol. 2020; 88: e70.
12. Py C, Martina M, Diaz-Quijada GA, Luk CC, Martinez D, Denhoff MW, et al. From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology. Front Pharmacol. 2011; 2: 51.
13. Kikuchi K, Sugiura M, Nishizawa-Harada C, Kimura T. The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system. Biotechnol Rep (Amst). 2015; 7: 17-23.
14. Chen CY, Tu TY, Jong DS, Wo AM. Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange. Biotechnol Bioeng. 2011; 108: 1395-403.
15. Schieder M, Rötzer K, Brüggemann A, Biel M, Wahl-Schott C. Planar patch clamp approach to characterize ionic currents from intact lysosomes. Sci Signal. 2010; 3: pl3.