Optical sensing in POCT: the contribution of the Institute of Applied Physics of the Italian CNR

Introduction

Over the past 20 years, there has been a growing demand from physicians for devices able to carry out rapid and reliable measurements of chemical and biochemical parameters near the patient’s bed. These devices should allow the rapid formulation of a reliable diagnosis and/or the quick choice of the most appropriate therapy, avoiding the use of centralized laboratory analyses which implies to wait for the results a period ranging from a few hours up to, sometimes, a whole day. These are the so-called point of care testing (POCT) devices that are becoming essential to the analysis of all those pathologies, where a rapid medical intervention is crucial to patient life [1]. Some devices are already available on the market, such as those for the measurement of biomarkers of infarction (e.g. troponin, myoglobin, D-dimer, BNP, CK-MB) which permit the measurement of the analytes in short time, generally in the range of 15–20 min [2]. It is evident that in the event of a possible infarction, saving time, even a few minutes, in performing the correct diagnosis may be essential for the patient’s survival.

It is important to take into account that the general attitude of doctors is to adopt solutions that can be easily tolerated by the patient and that introduce a minimal risk to their safety. From this point of view, non-invasive sensors are definitely preferable. On the other hand, for some applications, the need for continuous monitoring of biological compounds necessarily obliges the physicians to the use of invasive sensors and, in that case, very strict regulations must be met to fully guarantee patient safety.

Optical sensing [3] can definitely play an extremely important role in this area. In the case of invasive applications, optical fibers guarantee unique performance thanks to their geometric versatility, easy handling and high degree of miniaturization. Optical fiber catheters with a diameter of tens of microns and miniaturized optic probe up to a few microns allow doctors to reach human body locations unimaginable with sensors based on different technologies. Moreover, the absence of electrical contacts and the immunity from electromagnetic interferences confer a larger degree of safety with respect to different types of sensors.

Sometimes the determination of only one parameter is sufficient but it is important to emphasize that very often it is necessary to determine and monitor a set of different parameters. In this context, optical biochips can play a key role in the development of POCT instrumentation. An optical biochip can be considered as constituted by a matrix of biosensors individually interrogated. Due to its miniaturization, its low cost and large-scale potential automation, its use can lead to a more efficient analysis than the equipment currently available in the lab. Unlike genomics and proteomics, where thousands of detection points are simultaneously controlled by fluorescence scanner [4], in most POCT applications, there is a need to measure only a limited number of parameters in order to detect the correct pathology or to control the effects of the administered therapy.

The present paper provides a summary of the activities carried out at the Institute of Applied Physics “Nello Carrara” in the field of biosensors and optical biochips for clinical diagnostics.

Invasive sensors in the gastroesophageal apparatus

Optical fiber sensors for the 24-h monitoring of bile and pH in gastroesophageal and enterogastric refluxes and for the continuous measurement of carbon dioxide in the stomach in intensive care patients have been developed or are under development.

The method of detection of bile-containing reflux in the gastro-oesophageal apparatus is based on the detection of the presence of bilirubin, the main biliary pigment. Notwithstanding the complexity of the optical behavior of bile and of bilirubin, it is possible to assert that bilirubin is a good marker for the detection of bile. The measurement is performed by means of the insertion of an optical fibre bundle into the stomach (or esophagus). The bundle is connected with a portable optoelectronic unit capable of sending the light coming from two light emitting diodes to an optical probe positioned at the end of the fiber bundle; the optical fiber probe is actually a miniaturized spectrophotometric cell, of 3 mm external diameter. One LED emits blue light (emission centered at 470 nm) which is absorbed by bilirubin and the other one emits light in the green region (emission centered at 565 nm) and is utilized as reference. Bilirubin is only used as a marker, in order to measure the exposure time of the stomach/esophagus mucosa to the materials contained in the biliary reflux, which is the clinically-relevant parameter. Bile-containing refluxes can take place from the duodenum into the stomach and from the stomach into the esophagus. They are considered contributing factors to the development of several pathological conditions such as gastric ulcer, “chemical” gastritis, upper dyspeptic syndromes, and severe esophagitis. Under certain conditions, the enterogastric reflux may also increase the risk of gastric cancer. After its conception [5] and first clinical tests at the third Surgery Clinic of the Florence University [6], clinical validations were carried out at the Division of Gastroenterology of University of Alabama (USA) [7], at the Department of Surgery of the Klinikum Rechts der Isar Technische of the Technical University of Munich (Germany) [8] and at the Department of Surgery of the Royal Adelaide Hospital in Adelaide (Australia) [9]. The instrument, Bilitec 2000 (Figure 1) is produced by a small Italian company (Cecchi srl). It was initially marketed by the Swedish company Synectics Medical and subsequently by the Medtronic. In 2004, Medtronic left the market sector of the instrumentation and devices for the gastroesophageal apparatus and after a period of stalemate, since the end of 2010 the device is marketed by EBNeuro srl.

Figure 1:

The instrument Bilitec 2000 for the continuous monitoring of the bile-containing refluxes.

The possibility of measuring bile-containing reflux directly measuring the presence of bile provides complementary information with respect to the traditional pH-metry and the more recent impedentiometry. With the use of the optical fiber sensor, which is truly portable and capable of continuous monitoring, it has been possible to detect bile-containing refluxes correctly for 24-h, by adopting the same procedure utilized in pH-metry, a routine diagnostic test used by physicians since the 1980s for the analysis of gastro-esophageal reflux.

A novel optical pH sensor has been recently proposed (Figure 2, left), with the final purpose of its combination with the bile sensor in the same catheter: it is based on a novel configuration [10], constituted by two optical plastic fibers cut at their distal end with an angle larger than the total reflection angle in order to ensure the coupling between the two fibers. Controlled pore glasses (CPGs) carrying methyl red as acid base indicator are suitably fixed along the lateral surface of the two fibers. pH changes cause a modulation of the light intensity coupled from one fiber to the other fiber due to the changes of the absorption properties of the methyl red immobilized on the CPGs, as it can be seen from Figure 2 (right). The first clinical tests are scheduled at the end of the year.

Figure 2:

Left: drawing of the optical pH probe.

Right: absorption spectra at different pH values, collected by connecting the optical fibre probe to a spectrophotometer.

The last sensor developed for applications in the gastroesophageal apparatus is the sensor for the detection of gastric carbon dioxide, developed in collaboration with the Institute of Chemical Process Development and Control of the Joanneum Research (Graz, Austria) [11]. The possibility of correlating the increase of gastric CO₂ to early warnings of disturbances in the tissue perfusion can have an important impact, in intensive care units, on the survival of the patient, in whom for example an increase in the gastric CO₂ levels has been correlated to the impaired perfusion of the gastrointestinal region, as a consequence of shock syndrome [12, 13]. The detection principle is based on the absorption properties of a sensing layer containing cresol red, modulated by the change of the CO₂ partial pressure of carbon dioxide, which is immobilized at the end of a 600 μm optical fibre. The sensor was thoroughly characterized in laboratory and its performances were compared with those of Tonocap, the instrument based on gastric tonometry, which is the present method for detecting partial pressure of gastric carbon dioxide. Measurements carried out on intensive care patients at the Department of Critical Care of the University of Firenze (Italy) showed for the first time very fast changes of gastric pCO₂ not distinguishable by Tonocap, characterized by response time of the order of 10–12 min, which could not be correlated to tissue perfusion and the origin of which should be carefully investigated (Figure 3).

Figure 3:

Values of gastric pCO₂ measured with the optical fibre sensor and with Tonocap on an intensive care patient.

The values of the endtidal pCO₂ (EtCO₂) and of the arterial partial pressure of CO₂ (PaCO₂) are also shown.

Measurement of immunosuppressants in transplanted patients

The correct dosage of immunosuppressants in transplanted patients in the first days after the transplantation is essential for the patient life: if levels of immunosuppressive drugs are too low, there is a danger of organ rejection, but, if they are too high, the patient may be unable to fight off infections.

Current general practice in the monitoring of immunosuppressive drugs is the periodic administration of the drugs (for example, every 12 h) with a blood sample being taken prior to administration and drug concentration levels being evaluated in the laboratory before the next dose. This provides information on the trough level, which is the lowest concentration reached by a drug before the next dose is administered. Recent studies have shown that more accurate clinical indication is given by the area under the drug concentration time curve – the “Area Under the Curve” (AUC) – as this value is better correlated with both the efficiency and the side-effects of immunosuppressive therapy than the trough level [14, 15]. However, obtaining accurate information on drug levels from the AUC requires regular and frequent monitoring of the concentrations in the blood over a period of time and this is absolutely not trivial in intensive care units. It is also important to consider that what is presently measured is the total concentration of the immunosuppressants in the blood composed by the fraction accumulated in blood cells, the circulating fraction bound to proteins and the circulating free fraction. On the other hand, it is generally admitted that the circulating free fraction – of the order of the 2–8% of the total concentration – is mainly responsible for the pharmacological activity but also for the side effects.

A novel optical platform [16] for the frequent monitoring of the immunosuppressants in transplanted patients was developed within the European project NANODEM (http://nanodem.ifac.cnr.it/). The key elements are given by:

• the body interface constituted by a intravascular microdialysis catheter (MicroEye^®, Probe Scientific Ltd) which extracts continuously the blood dialysate;

• a novel chip constituted by ten microchannels (produced by microfluidic ChipShop (Jena, Germany) under our design where heterogeneous inhibition binding immunoassays take place for the simultaneous determination of three different immunosuppressants;

• a fluorescence-based optical platform for the chip interrogation.

Figure 4 shows: (left) the typical calibration curve achieved for tacrolimus [17] and (right) the sketch of the different modules of the optical platform implemented in the European project. With the developed device, it will be possible: (i) to measure the concentration of the free fraction of the immunosuppressants, since only this component is able to diffuse through the microdialysis membrane and (ii) to perform frequent measurements, every 30–40 min of the immunosuppressants leading to a determination of the AUC.

Figure 4:

Left: Calibration curve for the tacrolimus immunosuppressant.

Right: the sketch of the different modules of the optical platform implemented in the European NANODEM project.

Currently trials are being planned at the Klinikum rechts der Isar hospital of the Technical University of Munich to investigate the importance of the free fraction in patients after kidney transplantation. These trials will make use of the body interface module developed within the project to extract the dialysate and the concentration of cyclosporine A and mycophenolic acid in these samples will be measured with standard laboratory methods and with the new device.

POCT device for sepsis biomarkers

Sepsis in one of the pathologies for which the concept of POCT is more appropriate. As a matter of fact, it has been demonstrated that delays of only a few minutes until administration of an effective calculated antibiotic treatment in septic shock can lead to a significant increase of mortality [18] if the origin of the inflammatory process is bacterial. Therefore the fast discrimination between viral and bacterial sepsis and, in case, the immediate identification of the correct antibiotic therapy is fundamental for the patient survival. In the complex clinical picture of septic patients – a new definition of sepsis was provided in 2016 by the Society of Critical Care Medicine and the European Society of Intensive Care Medicine [19] – biomarkers can provide supportive and supplemental data to clinical assessment [20].

In this framework, in a concluded European project (CAREMAN), we developed a fluorescence-based optical platform for the simultaneous measurement of up to four different biomarkers (Figure 5) in serum or plasma samples; an optical chip with thirteen different channel produced by microfluidic ChipShop under our design is the heart of the instrument; on the surface of each channel, where the affinity reaction takes place, there is a sensing layer selective to a specific bioreceptor. This platform is under optimization within a presently running European project (Hemospec, http://www.hemospec.eu/) in order to be able to perform a complete assay in 30 min. The choice of the different biomarkers is depending on the continuous evolution of the clinical studies performed by physicians in the effort to identify the best biomarker panel for sepsis. In the CAREMAN project, our attention was focused on procalcitonin (PCT) [21], C-reactive protein (CRP) [22] and neopterin. In the HEMOSPEC project, attention is still focused on PCT and CRP whereas neopterin is replaced by interleukin-6 and soluble urokinase plasminogen activator receptor (suPAR), with suPAR being considered in the last years as an important prognostic biomarker for sepsis [23].

Figure 5:

The optical platform developed within the CAREMAN project for sepsis biomarkers.

The 13-channel optical chip is shown in picture, with the grey arrow indicating the location of the chip inside the instrument.

Conclusions

The prototypes above described and developed at our Institute show a small contribution to the importance of the POCT devices in clinical settings but clearly evidence how optics can play a fundamental role in this area. There is an enormous potentiality not completely exploited proven by a long list of laboratory prototypes described in literature and never transferred in a real clinical scenario. The crossing of this gap needs a continuous and strict cooperation between the scientists who develop the device coming from the different disciplines, ranging from chemistry, biochemistry, to optics and engineering, and the physicians starting just from the conception of the first idea in order to avoid the development of devices which will never have the possibility to work in a real clinical setting.