Ultrasensitive fluorescence immunosensor based on mesoporous silica and magnetic nanoparticles: Capture and release strategy

Highlights

• Ultra-sensitive sandwich type fluorescence immunosensor for hepatitis B virus surface antigen (HBsAg) reported.

• The immunosensor works on the basis of magnetic capturing and solvent stimulation releasement of encapsulated cargo.

• Fe₃O₄ MNP serves as probe collector site and MSN-Rh. B as triggered mesoporous fluorescent capsule.

Abstract

Herein we designed a novel, highly sensitive, simple and amplified fluorescence immunosensing strategy for hepatitis B virus surface antigen (HBV surface antigen) (HBsAg) as a model based on the construction of a sandwich type probe. The operation mechanism of this immunosensing strategy is implemented by capturing and then stimulation-based-releasing of entrapped dye in the fluorescent capsules. The proposed probe is made by the Fe₃O₄ magnetic nanoparticle (Fe₃O₄ MNP) as a probe collector site and the Rhodamine B loaded-mesoporous silica nanoparticle (MSN-Rh.B) as a fluorescent mesoporous capsule and signal amplifier site. Such a methodology is benefited, from the advantages of the high ability of MSNs to be used as a scaffold for efficient dye encapsulation and the magnetic nanoparticles as efficient biological carriers. Under optimal conditions, the fluorescence signal (The fluorescence of solutions was measured using a quartz fluorescence cell (PMT voltage:720, Ex wavelegth:540, Em wavelength:568, All measurements were carried out at room temperature) increased with the increment of HBsAg concentration in the linear dynamic range of 6.1 ag/ml to 0.012 ng/ml with a detection limit (LOD) of 5.7 ag/ml. The relative standard deviation, measured between the resulting fluorescence peaks was obtained by 6.0%.

Introduction

The liver as a vital organ processes nutrients, filters the blood, and fights infections. Hepatitis B is a viral infection that attacks the liver and can cause both acute and chronic diseases. There is still limited access to diagnosis and treatment of Hepatitis B virus (HBV). Therefore, by investigating early diagnostic methods, serious injuries can be prevented. The virus has a core with internal antigens (HBcAgs), with a lipoprotein coating that contains surface antigens (HBsAgs). These surface proteins are highly immunogenic and induce Anti-HBs antibodies [1], [2], [3]. Up to now, different methods have been proposed to detect HBsAg including enzyme-linked immunosorbent assay (ELISA), Real-time PCR, Electrochemical assay, Chemiluminescence assay, etc [4], [5], [6], [7], [8]. But these methods often involve complex equipment and processes, long-time analysis, and can’t detect very small quantities of the analyte. Therefore, the development of simple, fast, and sensitive sensors is of high importance. Hybrid (organic-inorganic) materials have made a considerable assistant to scientists and researchers to make further progress toward smarter methods. These materials are thermally and chemically stable and allow the use of a wide range of organic functional groups, as reaction centers or as absorbing agents [9], [10], [11]. What have been so much taken into consideration in this vast area in recent years are porous materials and the use of them to design the gate-like and or triggered scaffolds [12]. In gated/triggered nanochemistry, various materials such as polymers, nanoparticles, biomolecules have been used as capping agents, and the control of different external conditions such as pH, temperature, ionic strength, applied potential, and incident photon have been used for the stimulation [13], [14], [15], [16], [17]. Possessing features such as high internal area, easy synthesis, biocompatibility, flexibility versus surface modifications, colloidal stability, and many other features, mesoporous silica nanoparticles (MSNs) are a good option for the mineral base required for these compounds [18]. Porous silica particles were initially synthesized in 1999 for a different purpose, but because of their unique features, they were used for a variety of applications such as catalyst, sensors, isolation, energy, drug delivery, etc [19], [20], [21], [22]. Among the various MSNs, MCM-41 seems to be more practical owing to the more uniform distribution of particle size and the greater control over the hole's diameter. The interesting aspects of these particles are the effect of synthesis conditions, such as temperature, pH, and molar ratio, on their structure [23], [24], [25]. The direction of current detection methods tends to amplify very weak signals. In this regard, fluorescence is well adapted to the construction of novel biosensors and allows the use of a vast range of different optical tags. Recent reports have shown that amplified modern optical detection based on a combination of nucleic acids, aptamers, antibodies, enzymes with metal NPs, semiconductor NPs like quantum dots, have improved the sensitivity and accuracy [26]. But a fascinating and practical aspect of a strategy is its simplicity, while perfectly enhancing the signal. The advantages of hybrid materials have attracted much attention to their application in the development of amplified detection methods and gated MSNs have been used in different optical sensors/biosensors [27], [28], [29], [30]. Recently, usage of biomolecules such as antibodies and aptamers, as capping agents has been reported. He et al. designed an ATP-responsive controlled release system, by implementing a mesoporous silica nanoparticle capped with aptamers [31]. Oroval et al., used this system for fluorescence detection of α-thrombin and As(III) [32], [33]. There already reports of Rhodamine B encapsulation in mesoporous structures in the construction of fluorescence biosensors [34], [35]. Compared to these designs, MSN based-pH triggered systems create fewer complex structures with high efficiency. There are many reports on the applications of magnetic nanoparticles in different branches of chemistry. Until now, different analytical applications of MNPs such as the combination of magnetic nanoparticles and signal generator sites, using different biological molecules, including DNA, aptamers, antibodies, have been reported [36], [37], [38], [39]. In this work, the MCM-41 loaded by Rhodamine B, was used as a fluorescent capsule for signal amplification. The MSN-Rh.B capsule was modified with HBV antibodies(Ab) to form MSN-Rh.B/Ab-1. The HBV antibody surface-modified magnetic nanoparticle (Fe₃O₄ MNP/Ab-2) was used for collecting and separation of the fluorescent capsule throughout the sandwich complex formation in the presence of HBsAg. The greater the amount of analyte is, the greater the number of MSN capsules containing fluorescent pigments, will be collected with Fe₃O₄ MNP/Ab-2. This can be considered as one of the main reasons for the high amplification level in our probe. pH triggering and solvent stimulation are very simple and fast methods of pigment entrapment and releasing, respectively, to produce the corresponding signal. With the aim of an external magnet, Fe₃O₄ MNP/Ab-2 enable the efficient and easy collection of the formed MSN-Rh.B/Ab-1-HBsAg- Ab-2/Fe₃O₄ MNP (sandwich probe) without any filtration and centrifugation. The designed immunosensor offers a high level of sensitivity and selectivity for HBsAg.