Sensing HIV related protein using epitope imprinted hydrophilic polymer coated quartz crystal microbalance

Abstract

We have developed a biomimetic sensor for the detection of human immunodeficiency virus type 1 (HIV-1) related protein (glycoprotein 41, gp41) based on epitope imprinting technique. gp41 is the transmembrane protein of HIV-1 and plays an important role in membrane fusion between viruses and infected cells. It is an important index for determining the extent of HIV-1 disease progression and the efficacy of therapeutic intervention. In this work, dopamine was used as the functional monomer and polymerized on the surface of quartz crystal microbalance (QCM) chip in the presence of template, a synthetic peptide with 35 amino acid residues, analogous to residues 579–613 of the gp41. This process resulted in grafting a hydrophilic molecularly imprinted polymer (MIP) film on the QCM chip. QCM measurement showed that the resulting MIP film not only had a great affinity towards the template peptide, but also could bind the corresponding gp41 protein specifically. The dissociation constant (K_d) of MIP for the template peptide was calculated to be 3.17 nM through Scatchard analysis, which was similar to those of monoclonal antibodies. Direct detection of the gp41 was achieved quantitatively using the resulting MIP-based biomimetic sensor. The detection limit of gp41 was 2 ng/mL, which was comparable to the reported ELISA method. In addition, the practical analytical performance of the sensor was examined by evaluating the detection of gp41 in human urine samples with satisfactory results.

Introduction

The concept of molecularly imprinted polymers (MIPs) has a long history dating back to the early 1930s (Polyakov, 1931). MIPs are artificial made receptors with the ability to recognize and to specially bind the target molecule (Wulff and Sarhan, 1972, Wulff, 1995, Vlatakis et al., 1993). The synthesis of MIPs involves the formation of a complex of a target molecule (template) with one or more functional monomers though either covalent or noncovalent bonds followed by a polymerization reaction with cross-linking agent. Upon removal of the template, the binding sits are produced that are complementary to the template in shape, size, and the position of the functional groups. The stability, ease of preparation, and low cost of MIPs have led to their assessment as substitutes for antibodies or enzymes in sensors, catalysis, and separations (Haupt, 2003, Alexander et al., 2006, Ye and Mosbach, 2008, Ge and Turner, 2009). Although creating a MIP against small molecules (Wang et al., 2006, Gao et al., 2007, Riskin et al., 2008, Riskin et al., 2009, Tan et al., 2009, Bui et al., 2010, Stringer et al., 2010, Pernites et al., 2011, Li et al., 2011) or peptides (Hoshino et al., 2008, Zeng et al., 2010) is straightforward now, imprinting of large structures, such as proteins and other biomacromolecules, is still a challenge (Shi et al., 1999, Bossi et al., 2001, Bossi et al., 2007, Ge and Turner, 2008, Cutivet et al., 2009, He et al., 2010, Cai et al., 2010, Zhang et al., 2011). The major problem associated with the imprinting of proteins lies in their restricted mobility within highly cross-linked polymer networks and the poor efficiency in rebinding. Meanwhile, the molecular size, conformational flexibility and sensitivity to denaturation of proteins would also make them difficult for imprinting. Consequently, some approaches, such as surface imprinting (Li et al., 2006, Tatemichi et al., 2007, Nematollahzadeh et al., 2011) and epitope imprinting (Rachkov and Minour, 2000, Tai et al., 2005, Tai et al., 2010, Nishino et al., 2006), have been developed to overcome these problems.

Epitope imprinting is first demonstrated by Minoura and coworkers for peptide recognition (Rachkov and Minour, 2000). In this process, a fragment exposed on the epitope of the target macromolecular is used as template, the resultant MIP recognize not only the template but also the whole macromolecule. For example, in 2006, Shea and coworkers use the epitope-peptides of cytochrome C and bovine serum albumin as templates for creating macromolecular receptors for proteins (Nishino et al., 2006). Recently, Tai and coworkers developed a MIP-coated quartz crystal microbalance sensor for the dengue virus NS1 protein using epitope-mediated imprinting (Tai et al., 2010). Compared with traditional protein imprinting approaches, epitope imprinting has several advantages (Ge and Turner, 2008). Firstly, more specific and stronger interactions with a fragment or small part of the macromolecule can lower the non-specific binding and improve the affinity. Secondly, the polymer can not only recognize the template but also the entire protein and the operation procedures are easier. Thirdly, the short peptides as the epitopes for imprinting are low cost. However, little work of epitope imprinting has been conducted for detection of proteins in real human fluid samples.

HIV-1 glycoprotein 41 (HIV-1 gp41) is a protein that sits in the virus coat of human immunodeficiency virus type 1 (HIV-1), which is the causative agent of the acquired immunodeficiency syndrome (AIDS) (Contreras et al., 2001). It locates in the phospholipids bilayer and plays an important role in membrane fusion between viruses and target cells upon gp120 binding of CD4. After the viral attaching to cells, gp41 is thought to undergo a conformational change to mediate the fusion of the viral and target cell membranes (Dwyer et al., 2003, Burton et al., 2004, Buzón and Cladera, 2006). gp41 is an important protein that is not only relevant in fusion, but its activation process may provide novel strategies for vaccine and antiviral drug development (Chan et al., 1998). Therefore, developing a convenient, sensitive method for determination of gp41 is of great meaning in early clinical diagnosis and pathogenetic condition monitoring.

In this work, a MIP-coated quartz crystal microbalance (QCM) biomimetic sensor for the gp41 protein was fabricated by epitope-imprinting techniques. Dopamine was used as functional monomer and a synthetic peptide with 35 amino acid residues, analogous to residues 579–613 of the HIV-1 gp41 was used as the template. gp41 fragment 579–613 is a major immunodominant region that can be recognized by antibodies from approximately 98% of AIDS patients. Commonly known as a neurotransmitter, dopamine is also a small-molecule mimic of the adhesive proteins. Recently, Messersmith reported a simple but versatile surface modification approach in which self-polymerization of dopamine at weak alkaline pH produced an adherent polydopamine coating on a wide variety of materials including noble metals, oxides, semiconductors, and ceramics (Lee et al., 2007). The polydopamine films were found to be a versatile platform for further modification, leading to tailoring of the films for diverse functional uses (Lee et al., 2007). The detailed understanding of the polymerization mechanism of polydopamine was unknown so far. The prevalent view was that in alkaline conditions catechol was easily oxidized to quinone form, and the cross-linking was attributed to the reverse dismutation reaction between catechol and quinone form of dopamine molecule (Burzio and Waite, 2000, van der Leeden, 2005). Recently, polydopamine has been used successfully for molecular imprinting due to the high stability, hydrophilicity and biocompatibility (Ouyang et al., 2007, Ouyang et al., 2008, Zhou et al., 2010). However, there is no report about the application of polydopamine in epitope-imprinting. In this work, we demonstrated that the epitope-imprinting polydopamine film can not only recognize the template but also recognize the corresponding protein. Furthermore, this new method offers several advantages such as easy preparation, high stability and sensitivity, which would make this approach more practical in detection and separation of bio-samples.