Surface plasmon resonance (SPR) biosensors have become important research tools for the real-time detection of molecular interactions, especially without having to resort to labeling, and have been widely adopted both in basic research and in industry. SPR has been established as the standard method in label-free optical detection schemes and offers numerous advantages over alternative methods, including rapid and real-time detection, high sensitivity and low sample consumption , , .
Recently SPR was also used for the detection of miRNAs , , which are small, non-coding RNA molecules that can reduce the translation of target proteins by binding to the untranslated region of the mRNA. Therefore miRNAs are important regulation molecules of all biological functions, and alterations of the miRNA expression profile lead to interference of the homeostasis of organisms , . Misregulated miRNA signal pathways have been found for nearly all disease patterns like oncology , , cardiovascular , neurological  or immunological disorders . Distinctive miRNA profiles can be matched to specific disease patterns, qualifying them as excellent diagnostic markers , . Because of that the development of diagnostic and therapeutic strategies based on miRNAs is a rapidly expanding field in biomedical research.
A standalone and easy-to-use SPR biosensor developed by Fraunhofer IOF and Fraunhofer IWS has been successfully used for the rapid detection of different molecular interactions and assays , . Here we describe the application of a revised version of this biosensor with a new sensor chip/microfluidic hybrid for the specific and sensitive detection of miRNA-93 , utilizing a RNA-DNA hybrid antibody for primary signal amplification.
2 Material and methods
2.1 SPR biosensor
The SPR measurements were performed using a revised compact SPR biosensor based on an established Fraunhofer SPR platform (Figure 1) . This biosensor uses angularly resolved analysis of the quasi-monochromatic reflectivity when illuminated by light-emitting diodes with a wavelength of 810 nm. Up to three 1D arrays can be measured in parallel utilizing three light sources and a three-channel microfluidic. This biosensor is composed of the measurement device and disposable sensor chip/microfluidic hybrid system, consisting of the SPR chip and an on-chip-microfluidic. The temperatures of both the chip and the fluidic system are controlled by Peltier elements integrated into the biosensor. For the fluidic actuation we used a syringe pump.
2.2 Sensor chips and microfluidic
The sensor chips are disposable polymer chips (60 × 13 mm2) manufactured by use of injection molding (KDS, Germany). On top they are coated with a gold layer of 45 nm thickness produced via magnetron sputtering. Optical elements are integrated directly into the bottom of the chip allowing a simple, immersion free chip handling and coupling to the biosensors optical readout system.
After chip functionalization and immobilization of ligands the sensor chip is encapsulated with a top plate via snap-in elements. This top plate is manufactured employing two component injection molding, using a hard component for the housing and a soft component for the three microfluidic channels within. The closed sensor chip/microfluidic hybrid can then be handled without touching the chip surface and be placed inside of the biosensor for measurement.
2.3 Chip functionalization
For all experiments the chips were functionalized according to the following procedure. Initially the SPR chips were cleaned for 45 s with Gold Surface Cleaning solution (Sigma-Aldrich, Germany). After rinsing with deionized water and drying under nitrogen flux the chips were separated into four measurement areas and functionalized with two thiol-modified oligonucleotide probes (10 μmol/l in 60 mmol/l MgCl2) and two controls, polyethylene glycol (HO-PEG-SH, 10 μmol/l, Iris Biotech, Germany) and Protein A (1 mg/ml, Sigma-Aldrich, Germany), overnight at 4°C in a humid environment. Following another washing step with deionized water the chips were blocked with HS-PEG-OH (10 μmol/l) for 30 min at room temperature and rinsed again with deionized water.
The immobilized thiol-modified oligonucleotide probes for the hybridization experiments were LNA-93 (5′-CACGAACAGCACTTTG/iSp9//3ThioMC3-D-3′), a locked nucleic acid oligo which can hybridize to miRNA-93, and the two negative controls DNA-MM-31 (5′-ThioMC6-D/AGCCAAGATGGTGGCAGATCT-3′) and LNA-31 (AGCTATGCCAGCATCTTG/3ThioMC3-D/). MiRNA-93 (5′- CAAAGUGCUGUUCGUGCAGGUAG-3′) was ordered from Sigma-Aldrich (Germany), the RNA-DNA-hybrid antibody (ABIN1515114) from antibodies-online (Germany) and all LNA and DNA oligonucleotides from Exiqon (Denmark).
2.4 SPR measurements
All SPR measurements were executed using a flow speed of 2.49 μl/s and a temperature of 30°C for the flow cell and the preheating of the tubes. The running buffer and the buffer used for diluting was 60 mmol/l MgCl2.
At the start of the experiment the chip was rinsed with running buffer. After that the sample to be measured was injected into the flow cell with a volume of 100 μl and 25 μl were pumped 100 times back and forth over the sensor surface. Next the chip was washed again with running buffer before the subsequent injection of the next sample or antibody. For the analysis of the binding signals and the calculation of the standard deviation 100 parallel data spots on the chip were averaged for each of the four measurement areas.
3 Results and discussion
3.1 Direct detection of miRNA-93
The first experiments were performed to study the capacity of the SPR biosensor when detecting the hybridization of miRNA-93. For this a concentration series of miRNA-93 diluted in MgCl2 buffer (60 mmol/l) was measured sequentially on a chip, ranging from 10−11 mol/l to 10−6 mol/l (Figure 3). Before, between and at the end of the measurements the SPR chip was rinsed with running buffer. The chip was not regenerated in between the different concentrations. The results indicate a limit of detection for the hybridization of miRNA-93 to the LNA-93 probe of around 10 nmol/l.
3.2 Primary signal amplification with RNA-DNA antibody
In order to improve the limit of detection we used a RNA-DNA-hybrid antibody for primary signal amplification. This antibody does only bind to RNA-DNA double-strands present on the chip surface and so only gives a signal response after successful hybridization of miRNA-93 to the LNA probe.
Based on preliminary tests (not shown) a concentration of 10 μg/ml RNA-DNA-hybrid antibody is suitable for amplification and was chosen for injection. After measurement of miRNA-93 (10 nmol/l or 10 pmol/l) and washing with running buffer the solution containing the antibody was pumped through the sensor.
Figure 2 illustrates that, depending on the signal increase during the hybridization step, one can observe a corresponding specific sensor response during the amplification step and therefore a signal enhancement for the injection of the RNA-DNA-hybrid antibody. Note that the Protein A coated reference surface binds the amplification antibody directly via its Fc region and can be used as a positive control of the amplification step. According to the diagram in Figure 4 this approach lowers the limit of detection to a concentration of around 10 pmol/l miRNA-93.
4 Conclusion and outlook
Using immobilized LNA probes we could successfully implement an assay for the detection of miRNA-93 on our revised compact SPR biosensor platform. The limit of detection for the direct label-free hybridization of miRNA-93 is in the range of 10 nmol/l. The addition of an amplification step with a RNA-DNA-hybrid antibody enhances the specific signal recognition by three orders of magnitude and lowers the limit of detection to around 10 pmol/l miRNA.
In future works miRNA expression profiles will be addressed by exploiting the imaging features of the sensor system . Furthermore, this assay scheme will be utilized to detect miRNA-93 in lysates from cell cultures offering the possibility to characterize samples qualitatively. Other methods for the primary or even secondary signal amplification of the signal will be examined in order to further reduce the limit of detection.
The authors thank Thomas Schubert from KDS Radeberger Präzisions-Formen und Werkzeugbau GmbH for the fabrication and assistance with the sensor chip/microfluidic hybrids.
Research funding: This work has been funded under Grant “DEMOST” (13N12848) by the BMBF (German ministry of education and research). Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animal use.
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