Filter paper grafted with epoxide-based copolymer brushes for activation-free peptide nucleic acid conjugation and its application for colorimetric DNA detection
Graphical abstract
Introduction
DNA sequence analysis is crucially important for several biotechnological applications including clinical diagnosis, forensic identification as well as pathogen detection in food and agricultural products [[1], [2], [3], [4], [5], [6]]. The basic principle for DNA detection is generally based on the specific binding between the DNA target and its complementary nucleic acid probe according to the Watson-Crick base-pairing rules [7]. Peptide nucleic acid (PNA), firstly introduced by Nielsen and co-workers in 1991, is a synthetic DNA analogue having an uncharged peptide-like backbone [8]. The absence of electrostatic repulsion between the neutral PNA and the negatively charged DNA brings about a number of favorable DNA binding characteristics of PNA including high thermal stability, greater sequence specificity and mismatch discrimination sensitivity, and less salt-dependency. Among PNA variants being investigated thus far, a conformationally rigid pyrrolidinyl PNA derived from d-prolyl-2-aminocyclopentane carboxylic acid (acpc) backbones (acpcPNA) developed by Vilaivan et al. [9] has recently emerged as a potential and effective nucleic acid probe for DNA biosensor in that it can form a PNA⋅DNA duplex with even higher affinity and specificity than Nielsen’s original aminoethylglycyl (aeg) PNA. The success of using acpcPNA as probe for DNA sequence determination have been continuously demonstrated by many techniques including MALDI-TOF mass spectrometry [[10], [11], [12]], quartz crystal microbalance (QCM) [13], surface plasmon resonance (SPR) [14,15], and electrochemistry [[16], [17], [18], [19], [20]]. Unfortunately, most of which require advanced instruments that can only be performed in well-equipped laboratories. The development of highly sensitive and specific, yet simple and economical test kit/assays for DNA without the demand for sophisticated instruments, which are suitable for point-of-care usages still remains a challenge.
Ever since the debut of the first commercial paper strip [21] followed by the widely used lateral flow paper-based strip for pregnancy test [22], cellulosic paper has been increasingly attractive as a simple and affordable material for developing diagnostic assays [23]. The porosity inherited from its 3D structure accommodates several detection formats ranging from capillary flow, 2D-lateral flow to microfluidic devices. The function of the paper-based device can be further expanded via printing, coating or impregnation [[24], [25], [26], [27]]. The abundant presence of hydroxyl groups also makes chemical modification straightforward. Several paper-based DNA sensing platforms utilizing immobilized oligonucleotide probes have been implemented. Yu and coworkers [28] have used divinyl sulfone chemistry to covalently immobilize small molecules, proteins, and DNA onto cellulose paper. The ability to detect protein–carbohydrate, protein–glycoprotein interactions, and oligonucleotide hybridization demonstrate the potential of the biofunctionalized paper for point-of-care diagnosis. Araújo et al. [29] employed a bifunctional linker 1,4-phenylenediisothiocyanate (PDITC) prior to immobilization of amine-functionalized single-stranded DNA to cellulose paper. Upon hybridization to Cy3-labelled complementary DNA sequence, fluorescent detection can be achieved by naked eyes. Based on capillary transport vertically on the DNA-conjugated paper, the assay was capable of distinguishing fluorescent-tagged amplicons derived from canine and human genomic/mitochondrial DNA in forensic samples. Cellulose-based colorimetric assay for pathogen DNA detection has recently been developed by Saikrishnan and co-workers [30]. Hexanethiol-modified DNA was conjugated to tosylated microcrystalline cellulose. Signal amplification of the strip-based bioassay was performed via biotinylated DNA target in combination with streptavidin-horseradish peroxidase conjugate (SA-HRP) employing 3,5,3′,5′-tetramethylbenzidine as a substrate. A detection limit of 0.1 μM which corresponds to an absolute amount of 10 pmol was achieved.
Inspired by the above-mentioned studies, the first paper-based DNA sensing platform employing PNA instead of DNA as a probe has been developed in our group. The assay is based on the concept of “Dot blot hybridization” in which the filter paper grafted with quaternized poly(2-(dimethylamino)ethyl methacrylate) (QPDMAEMA) brushes was used as a positively-charged support that can selectively capture negatively charged DNA targets without non-specific adsorption of other analytes. After hybridization with a biotinylated acpcPNA probe, the presence of DNA can then be visualized by an enzyme-based colorimetric assay employing SA-HRP coupled with o-phenylenediamine (OPD) as a chromogenic substrate [31]. Although quite an impressive detection limit (10 fmol, equivalent to 1 μL of 10 nM of DNA) can be reached by naked eye detection, the technique has a limitation for detection of DNA mixtures because the DNA was adsorbed by the modified cellulose paper via a non-specific electrostatic interaction. There should most likely be binding competition by other negatively charged species, as well as by other non-related DNA sequences so that they may compete with the target by occupying the binding sites on the paper, thereby decreasing the amount of absorbed DNA target and reducing the sensitivity of the assay. To overcome such limitation, a second generation paper-based DNA sensing platform using acpcPNA probe was developed, which relied on direct immobilization of the acpcPNA probes on cellulose paper via divinyl sulfone chemistry. After hybridization with the DNA analyte through capillary action, a cationic dye that can electrostatically interact with the negatively charged backbone of DNA was applied in order to monitor the PNA-DNA binding. Although highly sequence specific DNA detection was demonstrated, only a modest detection limit at DNA concentration of 200 nM (50 μL) has been achieved [32].
In light of our and others’ success in using surface-grafted polymer brushes as platforms that can provide multiple active sites per surface area for sensing probe binding [[33], [34], [35], [36], [37]], it is envisioned that the performance of this paper-based DNA detection platform in terms of detection limit as well as the ability to suppress non-specific adsorption may be further improved if the paper is surface-functionalized with an appropriate polymer. Herein, filter paper grafted with poly[glycidyl methacrylate-ran-poly(ethylene glycol)methacrylate] (P(GMA-ran-PEGMA) via surface-initiated reversible addition fragmentation chain transfer polymerization (SI-RAFT) is introduced as a versatile platform for direct acpcPNA probe conjugation. By comparing with our previous work [31] where the positively charged QPDMAEMA brushes-modified filter paper had been used to detect DNA target via non-specific electrostatic interaction, this copolymeric system was chosen to generate multifunctional active layer for PNA binding due to the following advantages: (1) the epoxide group of the GMA unit can act as an active site for specific covalent coupling of the capture PNA probe via epoxide ring opening. Unlike diisothiocyanate or divinyl sulfone-modified surfaces, the epoxide ring is chemically stable, yet highly reactive towards nucleophilic attacks without the need for additional activation by coupling agent [33,[38], [39], [40], [41], [42], [43]]. (2) the PEGMA unit incorporated in the copolymer, as well as the hydroxyl group generated from the epoxide ring opening following the PNA probe immobilization, are hydrophilic and should prevent non-specific adsorption of non-target DNA as well as other non-DNA components that may complicate the detection. Detection of the captured DNA target was achieved by an enzyme-based colorimetric reaction via a sandwich-hybridization assay employing biotinylated acpcPNA probe (b-PNA) as a reporter probe together with SA-HRP conjugate and OPD substrate [[44], [45], [46]]. The working principle of the present paper-based DNA sequence determination platform is demonstrated in Scheme 1.
Section snippets
Materials
Whatman No. 1 filter paper was used as the membrane. PEGMA (98%), GMA (99%), dimethylformamide (DMF), 4,4-azobis(4-cyanovaleric acid) (ACVA), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPD), 4-(dimethylamino)pyridine (DMAP), N,N’-dicyclohexylcarbodiimide (DCC), o-phenylenediamine (OPD), bovine serum albumin (BSA), urea–hydrogen peroxide (urea–H2O2) and streptavidin–horseradish peroxidase conjugate (SA-HRP) were supplied by Aldrich (USA). PEGMA and GMA were purified through a column
Preparation and characterization of P(GMA-ran-PEGMA)-grafted filter paper and subsequent conjugation of capture PNA probe
The ACVA initiator was immobilized on the filter paper via esterification of the hydroxyl groups on the cellulose filter paper using DCC as a coupling agent in the presence of DMAP as catalyst. The P(GMA-ran-PEGMA) was then grafted onto the filter paper via a controlled SI-RAFT polymerization to ensure that the molecular weight and copolymer composition can be well-controlled. In this research, target degree of polymerization and copolymer composition (GMA:PEGMA) were set at 200 and 30:70,
Conclusions
In this work, we demonstrated the successful preparation of a P(GMA-ran-PEGMA)-grafted filter paper via SI-RAFT polymerization as characterized by FT-IR. The GMA part of the polymer provided a convenient reactive site for direct immobilization of a PNA probe for capturing a specific DNA sequence. The developed paper-based platform can be used for DNA sequence determination. It should be emphasized that this simple and versatile strategy based on epoxide ring-opening can be done under relatively
Acknowledgements
The financial support for this project was provided by Directed Basic Research Grant from the Thailand Research Fund (DBG5580003), the Distinguished Research Professor Grant from the Thailand Research Fund and Chulalongkorn University (DPG5780002).
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