Early Sepsis Diagnosis via Protein and miRNA Biomarkers using a novel Point-of-Care Photonic Biosensor

Sepsis is a condition characterized by a severe stage of blood-infection often leading to tissue damage, organ failure and finally death. Fast diagnosis and identification of the sepsis stage (sepsis, severe sepsis or septic shock) is critical for the patient's evolution and could help in defining the most adequate treatment in order to reduce its mortality. The combined detection of several biomarkers in a timely, specific and simultaneous way could ensure a more accurate diagnosis. We have designed a new optical point-of-care (POC) device based on a phase-sensitive interferometric biosensor with a label-free microarray configuration for potential high-throughput evaluation of specific sepsis biomarkers. The sensor chip, which relies on the use of metallic nanostructures, provides versatility in terms of biofunctionalization, allowing the efficient immobilization of different kind of receptors such as antibodies or oligonucleotides. We have focused on two structurally different types of biomarkers: proteins, including C-reactive protein (CRP) and Interleukin 6 (IL6), and miRNAs, using miRNA-16 as an example. Limits of Detection (LoD) of 18 μg mL-1, 88 μg mL-1 and 1 μM (6 μg mL-1) have been respectively obtained for CRP, IL6 and miRNA-16 in individual assays, with high accuracy and reproducibility. The multiplexing capabilities have also been assessed with the simultaneous analysis of both protein biomarkers.


Introduction
Sepsis is a condition expressed as a critical whole-body inflammatory response due to an infection. The first stage of sepsis involves a systemic inflammatory response (SIRS), followed by a severe sepsis which is characterized by organ dysfunction (liver, kidney, lung and heart). Further deterioration results in a sepsis shock that could cause hypotension and, ultimately, death [1]. Currently, sepsis is the main cause of death in Intensive Care Units (ICU) and its incidence is increasing worldwide with a mortality rate between 40 to 50% in developed countries [1][2][3][4]. The mortality rate increases with delayed diagnosis or with inappropriate antibiotic therapy, which implies, besides the human losses, in a high economic cost for the healthcare systems [4]. A well-timed and precise diagnosis of sepsis is really essential for the on-time selection of the most appropriate therapy.
Diagnosis of sepsis is not straightforward. There has been an extensive clinical research focused on identifying a reliable panel of biomarkers associated to sepsis.
Currently, there are more than 170 different biomarkers that could be useful for either its diagnosis or prognosis [4]. Some of them are already used at clinical level for diagnosis and treatment monitoring. Different pro-inflammatory cytokines (TNF, IL-1β and IL6) as well as C-reactive protein (CRP) and procalcitonin (PCT) have been proposed, among others. CRP is an acute phase protein, well established as a biomarker for infection and inflammation. High levels of CRP (40 to 200 µg mL -1 ) and PCT (above 0.5 ng mL -1 ) are known to be related with sepsis infection [5,6]. However, both biomarkers are also affected by other non-inflammatory processes, such as burns or traumas. Interleukin-6 (IL6), together with TNF-α and IL-1β, mediates the initial response of the innate immunity to injury or infection, enhancing the liver production of acute phase reactants, including CRP [1,5]. Additionally, circulating microRNAs (miRNAs) are acquiring importance as biomarkers for the non-invasive detection of diseases [7]. The evaluation of miRNAs differential expression levels in body fluids such as blood, saliva or urine can be a useful tool for diagnosis and prognosis. Some of them have been identified for playing a relevant role in the progression, diagnosis and staging of sepsis (i.e. miRNA15a, miRNA-146a, miRNA-16 or miRNA-223, among others) [8 -10].
Protein biomarkers like CRP and IL6 are individually analyzed following conventional techniques such as immunoassays or flow cytometry, which are precise and reliable, but they are also time consuming and require specialized personnel [11]. For the detection of circulating miRNAs, the most common techniques are real-time reversetranscription polymerase chain reaction (qRT-PCR), northern blot and fluorescent microarray technology. Due to the small size of the miRNAs and their low concentration in biological fluids (pM -fM) these techniques need a large amount of sample, and sometimes lack sensitivity or robustness and are time-consuming [8].
Combining the detection of several of the most relevant biomarkers would increase the overall sensitivity and specificity of sepsis detection. Adding also the identification and quantification of the etiological infectious agent would ensure a more accurate diagnosis, a timely start of the appropriate antibiotic treatment and the improvement of the outcome of the sepsis process. Therefore, having a technology capable of detecting all these targets in a multiplexed analysis in a prompt, specific and simultaneous way, ideally at the patient's bedside or the ICU, might enable proper stratification of SIRS and sepsis patients.
Optical biosensors are ideal candidates for this purpose. Due to their integration capabilities they are excellent analytical tools to move the analysis from centralized laboratories to the point-of-care. Their design often offers fast turnaround times, easyto-handle features and in some cases, simultaneous detection capabilities [12]. Several optical devices using microarray formats to perform multiplexed assays have been previously proposed. Some of them are based on conventional labelled strategies (i.e. fluorescence or chemiluminescence) but others follow label-free approaches as the reflectometric interference spectroscopy, surface plasmon resonance imaging or the arrayed imaging reflectometry (AIR) [13][14][15]. Kemmler et al. [15] proposed a POC device for sepsis diagnosis based on a microarray using Total Internal Reflection Fluorescence (TIRF). The POC was employed to evaluate several sepsis biomarkers but required fluorescent labels and involved several fluid handling steps such as dilution, mixing, separation, pre-incubation and incubation to carry on sandwich and inhibition assays. Internal calibration in the biochip was also needed to prevent inaccuracy. Also, Mace et al. [16] used their developed AIR technology to detect various cytokine proteins involved in the body inflammatory response. Specific antibody-based macroarrays were manually generated. The design of the device allowed visualization and tracking of up to 8 differentiated spots, which limited its multiplexing potential and produced a high variability among measurements.
Recently, we presented a novel optical phase-sensitive interferometric biosensor based on microarray configuration as a new platform for high-throughput evaluation with potential of detecting specific biomarkers in blood plasma [17,18]. This POC has been developed within the framework of a Horizon 2020 European project (RAIS, www.raisproject.eu). The device has a small compact size (20 x 14 x 23 cm), is portable and user-friendly, giving results in around 1 minute. It requires 1 cm 2 sensor chips and offers a large field of view (20 mm 2 ). This feature facilitates the generation of dense arrays, which could allocate thousands of spots to be visualized simultaneously. We here demonstrate for the first time the feasibility of this POC device for the detection of protein and miRNA sepsis biomarkers. We have selected CRP and IL6 as protein targets, and miRNA-16 as miRNA model, being one of the several miRNAs with suspected diagnostic value to sepsis. We have developed and optimized all the biofunctionalization protocols and individual detection assays for each of those biomarkers. Moreover, we also show the multiplexed potential for the simultaneous detection of CRP and IL6, demonstrating the capabilities of our POC device for fast and label-free diagnosis of several biomarkers.

Chemical and biological reagents
Monoclonal antibody against CRP named ab183 (anti-CRP) was produced at and were purchased from Sigma-Aldrich (Steinhem, Germany). Diethyl pyrocarbonate (DEPC), Protein G from Streptococcus sp. and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Steinhem, Germany). Oligo Analyzer software and RNA fold webserver was employed for secondary structure and self-annealing prediction of the probes and targets. DNA capture probes incorporating thiol (SH-DNA) at the 5'-end were purchased from IBIAN Technologies (Zaragoza, Spain) (see Table   1). Antibody against DNA/RNA duplex (Anti-DNA/RNA) was purchased from Kerafast (Boston, USA). All the buffers and other solutions for miRNA detection were prepared using DEPC-H2O (MilliQ water incubated overnight with 0.1% DEPC and autoclaved for 1h at 121 °C). All solid plastic and glass materials were autoclaved for 1h at 121 °C. The POC device (dimensions: 20 x 14 x 23 cm) has three main parts: a light source consisting of an external fiber-coupled LED 660 nm (Thorlabs), optical components (detailed in [17]), and an electronic assembly that controls the components and communicates with the computer. The LED light source was chosen to spectrally overlap with the transmission peak of the gold nanohole array (Au-NHA) used as plasmonic sensor chip [18]. The binding events occurring on the surface of Au-NHA chips were monitored with a custom designed LabView software which records the phase changes and renders an OPD (optical path difference) map image.

Spotting of Au-NHAs chips
All the spotting experiments carried out for the generation of the microarrays have been

Plasmonic Gold Nanohole Array chips
Fabrication of the plasmonic sensor chips was done in a clean room using wafer-scale and high-throughput fabrication methods. Fused silica wafers were coated with Ti/Au (10/120 nm) and patterned with 200 nm diameter and 600 nm period nanohole arrays.
The fabrication process is explained in detail in [18]. Before use, the Au-NHA chips

POC biosensor
The POC biosensor device has been previously described [17]. It is based on a lensfree interferometric microscopy which evaluates changes in the topography of transparent surfaces, allowing direct observation of target-binding events. Briefly, the device is an optical microarray reader with large-field-of-view (20 mm 2 ) (FOV), in which a polarized light beam is sheared into two beams (reference and signal, see Figure 1a)  (Figure 1c). In this format, each spot can eventually be related to a specific biomarker, allowing the immobilization of multiple bioreceptors to target different sepsis biomarkers.
To evaluate CRP and IL6 with the POC biosensor, we designed a direct and label-free immunoassay strategy, using antibodies as specific bioreceptors immobilized on the sensor chip surface. Direct assays are always the desired option, as they involve a simple one-step format, which reduces time and hands-on involvement, and facilitates further automation for POC deployment. In order to maximize the efficiency of the assay, the antibody should be as exposed as  The specificity provided by the chosen strategy was assessed with different controls as summarized in the Figure 3 for the CRP assay. We observed that the addition of the target protein resulted in a clear signal enhancement ((ΔOPD= 8.83, in Figure 3(1)).
However, no OPD increase was observed if the array was incubated with a different protein (in Figure 3(2)). Similarly, incubation of CRP over a non-specific antibody resulted in negligible increase in the OPD (ΔOPD= -1.47) (see Figure 3(3)). Moreover, in the absence of any antibody (i.e. only spotted Protein G arrays and BSA) the CRP did not bind (Figure 3(4) corroborating the lack of non-specific binding over the blocked surface. These different tests confirmed that the signal corresponds exclusively to the specific recognition of the protein for its specific antibody (in this case, CRP and anti-CRP, but extendable to any other antigen-antibody pair).
With the aforementioned conditions the corresponding calibration curves for both CRP and IL6 were obtained by incubating several arrays (8x8 spots) with different target concentrations (Figure 4a and c). The calibration curve for CRP showed a linear concentration-dependent region before reaching a saturation. A limit of detection (LoD) of 18 μg mL -1 was estimated. Since CRP levels in blood plasma of healthy individuals are commonly found below 10 µg mL -1 and can drastically increase to around 300 µg mL -1 in patients with severe infection [19], our approach can comfortably allow the detection of this protein in any infection particularly, sepsis. In the case of IL6, a linear response was observed without reaching saturation for the range of concentrations analyzed. Although it showed a good fitting (R 2 =0.9647) the LoD was relatively high (LoD= 88 µg mL -1 ) for the requirements for sepsis diagnosis. The affinity of the antibody might have an influence in this lower level of detectability compared for instance with CRP. However, this can be also associated to the much lower MW of IL6, since the working principle of the device is related to the refractive index changes on the surface, which in turn is related to the mass (as described in the experimental section and ref [17]).
In order to establish the accuracy of the method, some spiked blank samples for both biomarkers that fit within the linear range of the curve were prepared and analyzed with the biosensor.  Figure 5).

miRNA detection assay
In the case of miRNA assays, a complementary DNA probe to the target sequence was designed to specifically detect miRNA-16 via DNA/RNA hybridization. A thiol group was added to the 5'-end of the probe to facilitate the attachment to the gold surface through the formation of stable self-assembled monolayers (SAM) [20]. A poly thymine sequence with 15 thymines (T15) was introduced after the thiol group to move away the complementary part from the gold surface (see Table 1). The biofunctionalization process (Figure 6a) involved the spotting of the thiolated DNA probes to generate the array followed by a blocking step of the remaining gold area with BSA. In this case, given the relatively small size of the miRNA (MW= 6.6 kDa) an amplification step was considered by using an antibody that specifically binds only to DNA/RNA duplexes [21].
This anti-DNA/RNA antibody does not cross-react with double stranded DNA, double stranded RNA, or single DNA and RNA. The considerably high molecular weight of the antibodies (MW= 115 kDa) triggers a larger phase change, which in turn enhances the overall signal and improves the overall sensitivity (Figure 6b, c).
The immobilization steps showed similar behavior regardless of the DNA probe immobilized as can be seen in Figure 6d where similar signals were obtained for three different sequences (i.e. between 20-25 bases). The great affinity between the gold and thiol permits a simple, fast, direct one-step immobilization. As in the protein-based assay, the BSA blocking step also resulted in an OPD decrease consistent with its adsorption over gold (see Figure 6d). As can be seen in Figure 6c, With the conditions previously detailed, a calibration curve for miRNA-16 was obtained (Figure 7b). The curve shows the signal obtained in the amplification step with the anti-DNA/RNA. Also, in this case, a saturation region is observed for some of the miRNA concentrations analyzed. The LoD obtained was around 1 µM; this value is limited for the clinical requirements of this family of biomarkers, which commonly are within the nM -fM range in serum or plasma [8]. However, the developed assay using a sequence of DNA as bioreceptor offers a fast, straightforward label-free methodology for miRNA detection and illustrates the versatility of the POC device to measure not only proteins but also oligonucleotides.
In general terms, the sensitivity level could be further enhanced by additional amplification strategies which increased the overall mass attached to the surface.
Thus, for the protein biomarkers, the addition of a second antibody in a sandwichbased assay, either free or even conjugated to large entities such as nanoparticles could be a feasible and commonly used option. This would be also possible for the miRNA detection by directly coupling the ant-DNA/RNA antibody to the same kind of nanoparticles, which would eventually increase even more the signal.