High-performance portable graphene field-effect transistor device for detecting Gram-positive and -negative bacteria

https://doi.org/10.1016/j.bios.2020.112514Get rights and content

Highlights

  • Serious side-effects in the excess of antibiotics have been issued.

  • Nanotechnology to discriminate gram-positive and -negative bacteria are needed.

  • On-site high-performance bacteria detection by graphene electronics were demonstrated.

  • Novel interfacial chemicals for immobilizing bioreceptors on the graphene were synthesized.

  • Graphene portable devices at the on-site were designed and applied.

Abstract

Current techniques for Gram-typing and for diagnosing a pathogen at the early infection stage rely on Gram stains, cultures, Enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and gene microarrays, which are labor-intensive and time-consuming approaches. In addition, a delayed or imprecise diagnosis of clinical pathogenic bacteria leads to a life-threatening emergency or overuse of antibiotics and a high-rate occurrence of antimicrobial-resistance microbes. Herein, we report high-performance antibiotics (as bioprobes) conjugated graphene micropattern field-effect transistors (ABX-GMFETs) to facilitate on-site Gram-typing and help in the detection of the presence or absence of Gram-negative and -positive bacteria in the samples. The ABX-GMFET platform, which consists of recognition probes and GM transistors conjugated with novel interfacing chemical compounds, was integrated into the microfluidics to minimize the required human intervention and facilitate automation. The mechanism of binding of ABX-GMFET was based on a charge or chemical moiety interaction between the bioprobes and target bacteria. Subsequently, ABX-GMFETs exhibited unprecedented high sensitivity with a limit of detection (LOD) of 100 CFU/mL (1–9 CFU/mL), real-time target specificity.

Introduction

Antibiotic medications are very important for treating or preventing bacterial infections by killing or inhibiting the growth of bacteria. Antimicrobial drugs have been used to treat a series of pathogenic infections and are applied to a wide variety of host organisms in animals and humans. However, the misuse and overuse of antibiotics in humans and animals can lead to a serious problem concerning the spread of antibiotic-resistant bacteria which cause untreatable infections (Currie et al., 2014; Kennedy et al., 2008; Kiraly et al., 2016; Li et al., 2016). The major reason for the overuse of antibiotics is the insufficient clinical information differentiating between Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB) due to the absence of on-site rapid Gram typing or classifying technologies (Smith et al., 2018).

A technique, Gram statin, exists for Gram typing (Hu et al., 2018). Although this method is simple, easy to perform and highly accurate, the procedure is labor-intensive and time-consuming. Therefore, many biosensors for bacterial detection have been developed, such as colorimetric, cyclic voltammetry (CV), and fluorescence-based fluidic sensors. In particular, field-effect transistor (FET) methods have shown impressive results, namely, rapid and accurate bacterial detection (Moudgil et al., 2020; Wu et al., 2017). However, these detection methods also have critical limitations, including i) necessitating a time-consuming process for discrimination, ii) having low accuracy with aged bacteria samples and iii) requiring specific fluorescent readers (Moscicki et al., 1987; Ouyang et al., 2015; Romero et al., 1988). Therefore, the development of alternative methodologies, that are capable of rapid, real-time, sensitive, selective, and multiplexed detection remains a challenge.

To date, various bioprobes used in analytical methods, including enzyme-linked immunosorbent assay (ELISA), lateral flow assays (LFAs), CV and chemiresistive or impedimetric sensors (García-Aljaro et al., 2010; Singh et al., 2019; Ugolini et al., 2018; Vaisocherová-Lísalová et al., 2016; Wang et al., 2018), have been introduced owing to their high sensitivity and specificity in bacterial detection. However, these bioprobes are suitable for detecting only previously identified bacteria, making them unsuitable for real-time monitoring. Recently, several antibiotics, such as natural antimicrobial peptides (AMPs), β-lactams, polymyxins and glycopeptides, were presented as bioprobes because of their high sensitivity and selectivity in the detection of Gram positive/negative bacteria in a mixture of bacterial strain. In particular, AMPs and glycopeptides have been highlighted owing to their unique property with strong affinity to the surface of bacteria. Therefore, various types of AMPs and glycopeptides have been studied for developing high-performance sensors.

In this study, we first demonstrated a real-time, portable device, namely, a dual antibiotics (as bioprobes)-conjugated graphene micropattern field-effect transistor (ABX-GMFET) combined with a microfluidic chip for discriminating bacteria at an early stage. Specifically, the bioprobes for identifying clinical GNB and GPB were founded by vancomycin and magainin I owing to their high affinity toward GPB and GNB groups. In addition, the novel chemical linkers, bis(2-aminoethylene)perylene-3,4,9,10-tetracarboxyldiimide (PDA) and 1-pyrenebutyric acid N-hydroxysuccinimide ester (PANHS), were synthesized to improve the immobilization capability of bioreceptors on GMs, as verified by density functional theory (DFT) calculations and 3D structure modeling. The real-time responses of liquid-ion gated ABX-GMFETs toward various bacteria showed high sensitivity and selectivity, and their maximal responses were clearly observed depending on the affinity for the bacteria (the limit of detection (LOD) of both ABX-GMFETs is approximately 10°–101 CFU/mL). Moreover, the magainin I-conjugated GMFET (magainin I-GMFET) for GNB detection showed higher sensitivity than the vancomycin-conjugated GMFET (vancomycin-GMFET) for GPB detection with the same amount of bacteria. The Langmuir constants (K) were estimated by fitting the real-time responses and were used to evaluate the affinities for each species of bacteria. The high K values, 4.874 × 10−3 for GNB and 4.104 × 10−3 for GPB, indicate a strong intracellular affinity to the bioprobes. In addition, for the first time, an ABX-GMFET integrated into a dual-channel fluidic device showed high-resolution selectivity and sensitivity in the detection of bacteria from cultured samples, opening up the possibility of a portable ABX-GMFET system for on-site rapid bacterial Gram typing.

Section snippets

Materials and apparatus

4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), vancomycin hydrochloride, 1,5-diaminonaphthalene (DAN) and PANHS were purchased from Sigma Aldrich Co., Ltd. Antimicrobial peptide maganinin I was synthesized by Lusen Sci, and polydimethylsiloxane (PDMS) was purchased from Dow Corning. Poly (methylmethacrylate), 950 PMMA A4 4% in anisole, was purchased from MicroChem Co., Ltd., and bacteria such as clinical isolates Enterococcus faecium EFM 13-7-9104, Staphylococcus

Development of the dual ABX-GMFET microfluidic chip

The illustration in Fig. 1 presents the overall bacterial discrimination system based on the dual ABX-GMFETs integrated with a microfluidic chip, which consists of a PDMS microfluidic channel, a graphene transistor and electrodes (Fig. 1A, right side). The following bacteria-specific bioprobes were introduced onto the GM surface by interfacial reagents: i) vancomycin for GPB and ii) magainin I for GNB (Fig. 1B). Moreover, both the bioprobes on the GMFETs showed ultrahigh affinity, resulting in

Conclusions

In summary, for the first time, we presented sensitive and selective ABX-GMFET platforms combined with a fluidic chip for the dual detection of coexisting bacteria, namely, GPB and GNB. The ABX-GMFET achieved rapid detection of pathogenic bacteria by conjugation with specific bioprobes, such as vancomycin for GPB species and magainin I for GNB species. Specifically, the ABX-GMFET system was composed of MEMS processes and integrated with novel interfacial chemical compounds and Gram-positive or

Author contributions

O.S.K. and S.H.S. initiated the study. K.H.K. and S.J.P. performed the fabrication of sensing platforms, measured the real-time responses, characterizing the sensing electronics and demonstrated sensing mechanism. C.S.P. and J.K. synthesized interfacing chemicals and performed chemical analysis. S.E.S. and J.L. prepared experimental materials and analyzed the experiments. S.H.L. advised MEMS process to design the electronics. S.L., J.-S.K. and C.-M. R. incubated the bacteria and prepared target

CRediT authorship contribution statement

Kyung Ho Kim: Methodology, Validation, Writing - review & editing, Data curation. Seon Joo Park: Methodology, Validation, Writing - review & editing, Data curation. Chul Soon Park: Formal analysis. Sung Eun Seo: Formal analysis. Jiyeon Lee: Resources, Formal analysis. Jinyeong Kim: Resources, Formal analysis. Seung Hwan Lee: Methodology. Soohyun Lee: Resources. Jun-Seob Kim: Resources. Choong-Min Ryu: Resources. Dongeun Yong: Resources. Hyeonseok Yoon: Formal analysis. Hyun Seok Song:

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (318104-3); The National Research Foundation of Korea (NRF) Grant funded by the Ministry of Science and ICT for First-Mover Program for Accelerating Disruptive Technology Development (NRF-2018M3C1B9069834); The Cooperative Research Program for

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    1

    These authors contributed equally to this work.

    2

    Present Address: Department of Chemical and Biological Engineering, Hanbat National University, Daejeon 34158, Korea

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