Minimally Invasive and Volume-Metered Extraction of Interstitial Fluid: Bloodless Point-Of-Care Sampling for Bioanalyte Detection

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Minimally invasive and volume-metered extraction of interstitial fluid: bloodless point-of-care sampling for bioanalyte detection

Federico Ribet*a, Mikolaj Dobielewskia, Michael Böttcherb, Olof Beckc, Göran Stemmea, and Niclas Roxheda

(a) Division of Micro and Nanosystems, School of Electrical Engineering and Computer Science, KTH Royal Institute of Technology, Malvinas väg 10, 10044 Stockholm, Sweden. (b) MVZ Labor Dessau GmbH, Bauhuettenstrasse 6, D-06847 Dessau-Rosslau, Germany. (c) Department of Clinical Neuroscience, Karolinska Insitute, Stockholm, Sweden.

Abstract. Sampling of biological fluids is fundamental in health monitoring and, currently, blood analysis is the standard practice. However, blood sampling entails a series of drawbacks including pain, discomfort, and risk of infections. Interstitial fluid, having a good correlation with blood concentration dynamics for several analytes, is a promising alternative monitoring matrix because it can be sampled in a minimally invasively manner from the skin, without the need for the more invasive and painful extraction methods used for blood. Currently, there are no simple devices available to sample and store known amounts of interstitial fluid. In this work, a painless microneedle-based sampling device designed to extract 1 µL of fluid from the dermis was realized. The sampled liquid is metered and stored in a paper matrix embedded in a microfluidic chip. The sample can then be analyzed using state-of-the-art tools, such as mass spectrometry (LC-MS/MS).

1. Introduction techniques and no commercial solution available. Point-of-care sampling of biological fluids is To create a minimally invasive device to essential for patient monitoring and disease sample ISF, relevant for clinical use, at least six diagnosis. Even though blood sampling is key aspects have to be considered and currently the gold standard for therapeutic addressed: (i) achieve painless access to ISF to monitoring, it entails the use of invasive and limit patient discomfort, (ii) preserve simplicity painful methods, such as venipuncture and and contain the cost of all disposable parts of finger pricking. Alternative non-invasive options the device, (iii) limit the total sampling time, (iv) may consist in using other bodily fluids, such as sample ISF without significant contamination saliva, sweat or urine. However, the information from other specimen such as blood or sweat, (v) that can be collected in these specimens is ensure that the sampling is compatible with physiologically limited, compared to blood. In subsequent analyte measurements, and (vi) many cases, both the dynamics and the ensure volume metering of the extracted concentrations of several biomarkers and drugs sample to allow analyte quantification. are not strongly correlated or not yet fully Microneedles offer a minimally invasive understood [1], [2]. solution to penetrate the epidermis and provide Interstitial fluid (ISF) is an emerging access to the ISF in the dermal region [6]. Due to specimen for monitoring biomarkers and their size, microneedles are also painless both biomolecules. Dermal ISF is gaining particular during insertion and after removal, potentially interest because of the strong correlation with improving patient acceptance and compliance. blood dynamics and because it allows sampling By not drawing blood also the risks of infections, with minimally invasive techniques [3]–[5]. which are significant especially in point-of-care However, while the use of ISF as a measurement applications, are reduced [7], [8]. Moreover, the specimen has gained substantial interest, there use of microneedles has proven not to cause is currently a lack of satisfactory sampling

long term damage to the skin due to their certain applications is unquestionable, such an reduced size, providing a better solution for approach generally entails a series of challenges repeated sampling than collecting venous blood related to the range of detectable analytes and or performing finger pricks [9]. the reliability and quality control of the Current methods for ISF sampling are mostly measurement results. Therefore, a preferable based on vacuum-based suction [6], [10], [11], route for both versatile and reliable analysis of mechanically-applied overpressure [12], [13] or small sample volumes would be based on the absorbing microneedles [14]–[18]. Unlike for use of standard and state-of-the-art blood, an external pressure gradient needs to characterization tools, which can reliably be applied to extract the fluid from the skin provide simultaneous information on a broad tissue into a collecting device. Some of the main range of analytes from the same small volume issues with previous state-of-the-art devices are of sample. By this approach, also the medical related to their size and complexity, which validation and approval of the obtained results makes them practically difficult to implement as would be simplified. cost-effective disposable devices. Finally, to perform quantitative Another issue In ISF sampling is related to concentration analyses, a sufficient and specific the speed of extraction. As mentioned, since ISF amount of liquid must be sampled and stably is retained by the tissue, it does not stored. Previous ISF sampling methods did not spontaneously flow out from the skin after provide such functionality. pricking, inherently resulting in a slower Ultimately, the realization of a system that is extraction process compared to blood. While designed to take care of all steps from sampling the advantages of reduced invasiveness are to producing reliable results is of fundamental clear, the need for long waiting times might importance for the practical adoption of ISF as a hinder practical clinical use, as well as reduce measurement specimen. In this article, we the convenience for patients. To compete with present an interstitial fluid sampling device, blood sampling, the required time must be designed to extract a specific amount of ISF minimized and performed within a few minutes from within the skin of the human forearm, at maximum. without contamination by sweat. The device is Moreover, it is important to only sample ISF, based on a single stainless steel microneedle, without contamination from sweat or other which can be produced with conventional sources. [19] Some previously proposed methods, and that is connected to a simple solutions, e.g. based on blotting from the skin microfluidic chip. This laminated microfluidic surface after pricking, are prone to errors due to chip can collect and meter ~ 1 µL of ISF in contamination [20], [21]. Other alternatives, approximately 5 minutes. The sampled liquid is based on blistering, were shown to provide therein stably stored in a DBS paper matrix, altered results [22]–[24]. ready for subsequent off-line analysis. The The sampled fluid would then ideally be collected ISF can then be analyzed using state- stored within the sampling device so that it can of-the-art medical laboratory tools, such as be shipped to a laboratory in a stable form and liquid chromatography-tandem mass be analyzed. Currently, for blood analysis, the spectrometry (LC-MS/MS). The realized storage of blood in paper in the form of dried disposable sampling device is simple, compact, blood spots (DBS) is gaining interest and has and enables painless and potentially cost- great potential to be used in clinical practice in effective point-of-care sampling of metered different fields [25], [26]. Some blood amounts of interstitial fluid for streamlined microsampling devices were previously detection of a broad range of bioanalytes. proposed [27], [28], even providing in-situ measurement functionalities [29]. While the convenience of point-of-care direct readouts for

2. Design of the device the volume metering of the sampled ISF, determined by the paper dimensions. Figure 1a illustrates the sampling principle, with the designed system applied to the forearm. The system is composed of two parts: (i) a reusable pressuring inserter and (ii) a small disposable sampling device. The pressuring inserter (Fig. 1a) is responsible for the correct transdermal insertion of the microneedle and for producing a lateral radial overpressure guiding the ISF into the sampling device. The application of a certain pressure, radially converging toward the needle, is required to overcome the physiological ISF retention exercised by the skin. These two functions can be combined into a single device or be separated. Both solutions were tested in sampling experiments. The disposable sampling device is based on the integration of a hydrophilic stainless steel hollow microneedle, for intradermal skin penetration, and a microfluidic chip, for sample collection and storage (Fig. 1b). The use of microneedles obtained from standard stainless steel hypodermic needles, instead of e.g. silicon microneedles, is important to limit the cost of the one-time-use component. Importantly, the designed shape of the needles would allow the use of standard manufacturing processes for needle cutting and shaping. The side opposite to the tip of the microneedle is assembled into a microfluidic channel where a porous paper matrix is placed and arranged to be in contact with the open wall of the needle. The paper fibers bridge the liquid flow, from the vertically- placed needle lumen into the horizontal microfluidic chip, absorbing the liquid by capillary action. To verify the complete filling of Fig. 1. (a) Illustration of the system applied to the the paper matrix, a color indicator is used. The forearm. The system is composed of a sampling chip fluid flow in the paper dissolves a colored and an inserter, which is also responsible for applying powder dye present in the channel and, when an overpressure to the skin on a ring surrounding the the colored fluid is visible at the dedicated chip. (b) Illustration of the cross-section of the observation window (Fig. 1c), the device can be system. The ring applies pressure to the skin, guiding removed from the skin and stored for the ISF into the microneedle lumen and into the chip subsequent analysis. The geometry of the paper where it is absorbed by a paper matrix. (c) Illustration strip ensures that all its volume is filled before of the top view of the sampling chip. A colored dye in reaching such a location. This feature enables the paper allows visualization of the liquid front. Sampling is stopped when the liquid reaches the visualization window.

3. Materials and methods AM4113ZTL Dino-Lite, AnMo Electronics Corporation, Taiwan). Fabrication Analyte detection was tested with LC- The microneedles were realized by MS/MS using caffeine as an example of a target processing standard hypodermic needles to molecule. For detecting caffeine, similar LC- obtain 1-mm-long microneedles (1.9-mm-long MS/MS protocols were previously developed for overall, considering the portion assembled into analysis in whole blood from DBS paper. [30] the microfluidic chip) with the design illustrated For the presented measurements, 1 μL aliquots in Fig. 1b. The 32 G stainless steel needles (Novo of dyed artificial ISF with the addition of caffeine Nordisk A/S, Denmark) were processed using a at concentrations varying from 0.1 μg/mL to 9 femtosecond laser (Spirit, Spectra-Physics, MKS μg/mL were pipetted into different paper Instruments, USA). The microneedle surface was pieces, in duplicates. Samples were then let dry cleaned in a 10% citric acid solution in DI water into the paper matrix and stored. The dried and sonicated, resulting in hydrophilic surfaces. samples were then eluted and characterized The microfluidic chip base, in which the using LC-MS/MS (at MVZ Labor Dessau GmbH, microneedle is vertically assembled, was made Germany). of PMMA. The top part was made of two 170- Finally, to evaluate the penetration depth μm-thick layers of a double-adhesive tape (Tesa and the effect of microneedle penetration into SE, Germany) and a plastic foil (PP2280, 3M, the skin, an optical coherence tomography tool Sweden). 340-μm-thick paper pieces (Grade (OQ Labscope 2.0, Lumedica Inc., USA) was 238, Ahlstrom-Munksjö, Finland) were used. processed to obtain a geometry able to contain ~ 1 μL of liquid, with the shape illustrated in Fig. In-vivo studies 1c. Few μg of dry blue dye powder (Patent Blue In-vivo studies were performed on healthy VF, Sigma-Aldrich, Sweden) were poured on the human volunteers. The chosen sampling paper matrix to enable visualization of the front location was the inner forearm, because easily of the ISF flow during sampling. The desired accessible in most circumstances. Moreover, the geometries were patterned into the various thickness of the forearm skin layers allows materials using a CO2 laser (VSL 2.3, Universal access to the dermal region, rich of Laser Systems Inc., USA) and were then homogeneously distributed ISF with high laminated and assembled. correlation with concentration dynamics in Characterization methods blood, using 1-mm-long microneedles [31], [32]. The sampling continued until the indicator was The metering accuracy was evaluated by visualized by the operator unless the volunteer pumping an artificial ISF solution (phosphate decided to interrupt the procedure earlier. buffered saline solution, glucose, bovine serum albumin, dye) into the microneedle lumen at 4. Results and discussion defined flow rates, which were varied between the different runs. At the time in which the The assembled sampling device is shown in Fig. liquid crossed the line, corresponding to the 2a. The disposable part is compact and, because visualization window (c.f. Fig. 1c), the flow was of the materials and manufacturing stopped and the total amount dispensed by the technologies potentially usable, cost-effective. tool was recorded. For these tests, a syringe The microneedle size results in painless pump (NE-1002X Programmable Microfluidics insertion and its size is compared side-to-side with a commercial lancet for finger pricking in Syringe Pump, Pump Systems Inc., USA) was Fig. 2b. An enlarged picture of the microneedle used. The experiments were video recorded using a portable microscope camera (43- is shown in Fig. 2c.

In-vivo sampling Sampling was performed by applying the device to the inner forearm. The spring-loaded inserter and a pressuring ring were used to achieve insertion and apply a radial pressure of ~ 10 N to lead dermal ISF to the microneedle opening, respectively. The process was stopped when the liquid front reached the indicator location, i.e. the edge of the visualization window illustrated in Fig. 1c. The sampling time varied between 3 and 8 minutes (n = 15) between different tests and individuals, with an average of 5 minutes. Fig. 3a-d shows the filling of the paper matrix with ISF over time. In this case, complete filling was achieved in 3.2 minutes.

Fig. 2. (a) Sampling device placed on a fingertip, to highlight its small size. (b) Side-to-side comparison between a commercial lancet for finger prick and a sampling device, featuring a smaller needle. (c) Zoomed-in picture of an assembled stainless steel microneedle.

Volume-metering reliability

The volume-metering reliability was assessed by Fig. 3. (a, b, c, d) In-vivo sampling process overtime pumping an ISF surrogate solution into the after 20 s, 80 s, 130 s, and 190 s, respectively. The device at known flow rates it reached the blue liquid front is visible due to the presence of a visualization window. The minimum and soluble blue dye powder on the paper surface. For maximum flow rates used were 0.13 μL/min and visualization purposes, the entire paper matrix is 0.34 μL/min, respectively. These values visible in this device through a transparent cover and correspond to the fastest and slowest sampling not only through a visualization window at the end of rates observed during in-vivo experiments. By the filling path. imposing a flow rate of 0.13 μL/min, the OCT and skin recovery indicator location was reached after dispensing 1.06 µL, while with 0.34 μL/min, 1.13 µL were The insertion speed and the needle geometry dispensed. Therefore the difference between play fundamental roles in the skin penetration the minimum and maximum flow rates was process [33], due to the elastic properties of the within ±3.5%, with an average collected volume skin [34]. Fig. 4a shows an OCT image of the of ~ 1.1 µL. needle inserted in the skin. The skin bending around the needle is approximately 150 µm.

The needle opening, starting 250 µm below the

device base, is thus completely inserted inside The microneedle insertion was reported as the skin. Fig. 4b shows the pricking location painless by all individuals. Fig. 5a-d shows after device removal. After one hour, the pictures of the healing process of the skin after pricking location could not be visualized using the sampling procedure, over five hours. the OCT tool. Initially, signs of the skin pricking and the ring pressure are visible at and around the sampling location. The skin recovered within five hours (Fig. 5d). No permanent mark was visible days after the experiments. Detection of caffeine To verify the possibility to measure relevant physiological concentrations of analytes in small sample volumes, the concentration of caffeine in 1 µL spiked ISF surrogate was measured. The different concentrations in the various samples, processed as described in the previous section, were measured using LC-MS/MS. Fig. 6 shows the correlation between eluted samples and initial concentrations. Below the initial concentration of 2 μg/mL the measurements were considered unreliable and discarded. Fig. 4. OCT images showing in-vivo forearm skin cross-sections. (a) Microneedle, attached to a PMMA These results are preliminary and further base, inserted in the skin to visualize the skin investigations are ongoing to minimize this limit curvature around the insertion location. (b) Skin of quantification since by using LC-MS/MS imaged immediately after the device removal, significantly lower analyte concentrations can showing the pricking location. Colored lines were potentially be detected [35]. Moreover, the added to the images to highlight the different parts. characterization of real ISF samples is ongoing.

Fig. 5. Recovery process of the skin over time after the sampling procedure. Immediately after a device Fig. 6. Detected concentration in artificial samples removal, signs of the skin pricking and the applied eluted from paper and injected in the LC-MS/MS tool ring pressure were visible. Pictures were taken after against initial concentration values, as pipetted in the (a) 1 minute, (b) 25 minutes, (c) 2 hours, and (d) 5 paper matrix (1 μL), in duplicates per each hours, respectively. After 5 hours, marks are barely concentration step. The dashed line is the linear fit of visible. the data.

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