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# A toolkit of thread-based microfluidics, sensors, and electronics for 3D tissue embedding for medical diagnostics

## Abstract

Threads, traditionally used in the apparel industry, have recently emerged as a promising material for the creation of tissue constructs and biomedical implants for organ replacement and repair. The wicking property and flexibility of threads also make them promising candidates for the creation of three-dimensional (3D) microfluidic circuits. In this paper, we report on thread-based microfluidic networks that interface intimately with biological tissues in three dimensions. We have also developed a suite of physical and chemical sensors integrated with microfluidic networks to monitor physiochemical tissue properties, all made from thread, for direct integration with tissues toward the realization of a thread-based diagnostic device (TDD) platform. The physical and chemical sensors are fabricated from nanomaterial-infused conductive threads and are connected to electronic circuitry using thread-based flexible interconnects for readout, signal conditioning, and wireless transmission. To demonstrate the suite of integrated sensors, we utilized TDD platforms to measure strain, as well as gastric and subcutaneous pH in vitro and in vivo.

## Introduction

Implantable diagnostic devices (IDDs) and smart wearable systems (SWSs) that are capable of in situ sample collection and integration with the complex three-dimensional (3D) structure of biological tissues offer significant opportunities for diagnosing and treating diseases. Recent technological advances in the miniaturization of sensors and the fabrication of smart materials have progressively changed the landscape of health care by providing the ability to develop devices that can continuously monitor a patient’s health status15. Examples of such devices are electrocardiogram electrodes, temperature sensors, pH sensors, and flexible batteries that have been used for real-time health monitoring612. The development of such devices requires overcoming the challenges associated with the mismatch between the mechanical and topographical properties of the semiconductor-based electronics and the biological tissues. Flexibility and biocompatibility are other key characteristics needed for devices in these applications.

Materials such as polyimide13 and parylene14 have been extensively used as substrates for IDDs and SWSs. However, device microfabrication on such materials is expensive because it requires clean room facilities and specialized processing. Paper has emerged as a promising substrate for implantable devices and wearable electronics because of its universal availability, low cost, environmental friendliness, and ease of fabrication1517. Several paper-based platforms have been developed recently, such as lateral flow immunoassays for the detection of analytes18,19, detection of DNA or proteins20, and electrochemical biosensing21,22. Recently, nanofibrous polymeric substrates have been developed to fabricate elastic and flexible electronics that can be sutured3. The fibrous microstructure of the substrate enables gas and liquid permeation and promotes the ability to pattern metallic electrodes on them.

Although these substrates hold great promise for the creation of wearable and implantable devices, their overall structure and form has essentially remained two-dimensional, limiting their function to tissue surfaces, such as skin. However, the ability to integrate functional components, such as sensors, actuators, and electronics, in a way that they can penetrate multiple layers of tissues in a 3D topology would be a significant advance. For example, wound fractures and orthopedic implants, which have complex 3D structures, would greatly benefit from the implantation of physical (for example, strain) and chemical (for example, pH) sensors that can monitor the local tissue environment and provide valuable information to optimize patient-specific treatments. IDDs and SWSs can also benefit from a system that enables the delivery of bodily fluids from different parts of the same tissue or from different tissues to the sensing elements. Such a system for acquiring complex-sensing information distributed in space and time can provide more useful information about the function of an individual organ. Microfluidic systems are promising platforms that allow the manipulation of minute amounts of liquid (~nl) in small footprints23. The ability to fashion microfluidic networks for spatio-temporal chemical analysis in a tissue or organ environment in 3D would represent a significant advance. Microfluidic systems with integrated sensors have been widely used to perform molecular assays on blood samples24, capture circulating tumor cells25, and detect cancer-specific biomarkers26. However, these systems are mainly limited to planar structures and are able to transport liquid over small distances only.

From the fabrication standpoint, photolithography, screen-printing, and stamping methods are common approaches for the implementation of electrodes on planar substrates, which have also been used to realize functional sensing ‘tattoos’ on the body2,5,31. In these methods, the size of the electrodes depends on the feature size on the photo mask, stencil or shadow mask. When working with threads, the fundamental limit is set by the thread diameter, which has already reached sub-micron dimensions using conventional threading processes. The micropatterning of such threads using current textile technology, such as sewing, knitting, and embroidering to make functional devices justifies the scalability of thread-based devices for different sizes and shapes for various applications.

Our TDD is based on the use of functional threads as building blocks, which are inherently flexible and can be fashioned on any flexible substrate or sutured into any biological tissues in an arbitrary 3D geometric form. We propose the fabrication of threads with different physical, chemical, and biological functions to serve as sensors, microfluidics, and electronics, and to be integrated as a TDD (Figure 1). Hydrophilic threads were embroidered onto a highly hydrophobic woven fabric to serve as microfluidic channels for the controlled delivery of bodily fluids to the sensing zones. Conductive threads infused with nanomaterials, such as carbon nanotubes (CNTs), carbon nanopowders, polyaniline (PANI) and their combination, were used as thread-based electrodes for the in vitro and in vivo measurement of glucose, pH, temperature, and strain as representative physiological properties. Outputs of the sensors were connected to readout electronics on a different layer, which consisted of electronics for signal processing and wireless communication to a smartphone or a computer using conductive threads as interconnects.

Although prior studies regarding thread as a substrate focused exclusively on one aspect, such as microfluidics or sensors29,32 with a single thread type for ex vivo low-cost diagnostic applications, we demonstrate an integrated TDD platform for implantable applications with a diverse and greatly expanded toolkit of sensors, electronics, and microfluidic functions, such as custom-designed nano-infused threads as multiple chemical and physical sensors.

## Materials and methods

Phosphate-buffered saline solution (PBS), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were purchased from Sigma Aldrich, MA, USA. For preparation of the electrodes, carbon ink (E3456 Graphite Erconinc, MA, USA), silver/silver chloride ink (AGCL-675C, Conductive Compound, Hudson, NH, USA), functionalized CNTs (Sigma Aldrich), and PANI emeraldine base (Sigma Aldrich) were obtained. Polydimethylsiloxane (PDMS; Sigma Aldrich) and blue insulator (E6165, Erconinc, MA, USA) were utilized as dielectric covers of the electrodes.

### Doping of the PANI

The PANI was doped based on a previously published procedure33. A PANI base (500 mg) was added to 20 ml of HCl (0.1 M). The solution was mixed for 5 h at −4 °C inside an ice bath. The PANI-covered thread was kept in a desiccator before usage.

### CNT ink

CNTs functionalized with a carboxylic group were purchased from Sigma Aldrich. The CNTs were first premixed in IPA (2 mg ml−1) and stirred by a vortex, and then they were homogenized in an ultrasonic bath.

### Scanning electron microscopy

Scanning electron microscopy (SEM) images were acquired using FESEM ultra55 (12 kV). The microparticles were dried on a paper substrate, mounted on aluminum stubs using conductive carbon paint and sputtered for SEM analysis.

### Characterization of sensors

Characterization of the strain sensor was performed using a micro Instron 5542 mechanical tester (Norwood, MA, USA) with a 1-kN load cell. The two ends of the thread with a length of 15 mm were connected to moving stages, and uniform strain was applied, whereas the resistance was measured using a source meter (Keithley source meter 2400). Data were acquired after the output data were stabilized.

The pH- and temperature-sensing measurements were conducted using a customized potentiometric board using off-the-shelf components, including an LMP91200 as a main front-end amplifier component. For the pH readout, the voltage generated after transmission through an ultralow noise buffer was sampled and digitized by a microcontroller and then transmitted to an XBee LilyPad or Bluetooth low-energy (BLE) wireless module, and the data were collected in real-time on a smartphone or computer. The BLE package for cell phone communication is shown in Supplementary Figure S9.

Glucose sensing measurements were also performed utilizing a customized printed circuit board with off-the-shelf components, including a TI LMP91000 as a main component of the low-power potentiostat. The LMP91000 applied a voltage of 0.5 V to the glucose sensor, taking the current from the glucose sensor as input and generating analog voltage as an output. The output voltage, similar to the pH readout after sampling and digitization using the microcontroller, was sent to a wireless transmitter.

### In vivo measurement and animal protocol

The research protocol was approved and in compliance with Tufts University’s Institutional Animal Care and Use Committee (IACUC, protocol #M2013-53) in accordance with the Office of Laboratory Animal Welfare at the National Institutes of Health. Briefly, animals were anesthetized using 2–3% isoflurane inhalation, and the incision site was shaved and cleaned with alternating povidone–iodine and ethanol scrubs. For subcutaneous pH measurements, a small incision (<1 cm) was made, and the probes were inserted through the wound. Alternatively, two 18-G needles were inserted under the skin, and the probes were fed through them, ensuring that the ends of the two probes did not touch each other. For gastric pH measurement, the probes were fed through two 16-G oral gavage needles (Braintree Scientific, Braintree, MA, USA) and inserted through the mouth into the stomach. Last, for pH measurements of the blood, the animals were sacrificed via thoracotomy while under anesthesia, and blood samples were taken from the heart and placed into green-top blood collection tubes containing lithium heparin (Becton Dickinson).

## Results and discussion

### Fabrication of functional threads

Physical and chemical sensors, a microfluidic network, and interconnects are three components of the envisioned 3D TDD platform for intimate tissue integration. Functional threads, such conductive threads, nano-infused threads and hydrophilic threads, serve as building blocks for the realization of these components.

Electrodes, which are the key constituents of any chemical and physical sensors, were fabricated by sequentially passing the core cotton threads through multiple wells containing conductive inks (Figure 2a). Such a device was previously developed by our group to coat cell-laden hydrogels on suturing threads34. Conductive inks explored in this work include silver/silver chloride, carbon, CNTs, and PANI prepared as described in the experimental section (Figures 2b–h). A dryer was utilized to cure the coating layer on the thread whenever needed. In addition, an ultraviolet light was used for sterilization in animal studies. An image of the setup is shown in Supplementary Figure S1, and colorful dyes are added for better visualization. Meters of functionalized threads were fabricated using the proposed manufacturing method and collected on rotating spools (Figures 2b, c, and f).

For strain sensors, nano-infused threads were made by coating CNT and PDMS layers on elastic threads (polyurethane (PU) threads). The use of PDMS enhanced the mechanical integrity of the conductive layer and reduced the occurrence of delamination. For pH sensors, the working electrode was made from nano-infused threads coated with carbon and PANI, and the reference electrode was made from silver/silver chloride (Supplementary Figure S2). PANI was chosen because of its biocompatibility, high electrical conductivity, and superior stability in electrolytes35. Because it has acid/base-dependent oxidation states, it is an ideal material for pH sensors. Moreover, PANI forms a thin layer with a 3D network of interconnected nanofibrils (Figure 2d) that promotes its mechanical flexibility and enhances the mechanical integrity of the coated layer. Carbon is selected because it enhances the electrochemical reactivity and promotes electron transfer due to its unique physical and chemical structures36. Silver/silver chloride is a very common reference electrode for stabilizing the voltage level of the analyte solution. More conductive and dielectric threads are shown in Supplementary Figure S2.

The biocompatibility of the carbon/PANI, silver/silver chloride, and nafion utilized for the fabrication of chemical sensors has already been demonstrated in prior work37. Physical sensors were covered with layers of PDMS, which is a well-known biocompatible material. Cotton and polyurethane threads have also been shown to be biocompatible in previous studies38. Moreover, we also performed additional experiments to test the biocompatibility of cotton thread by culturing 3T3 cells (Supplementary Figure S3). This demonstrated that cotton thread has excellent cell viability even after 7 days.

To demonstrate the compatibility of the fabricated conductive threads with current textile technologies, a programmable embroidering machine (Brother PE-500) was used to pattern conductive threads on a woolen fabric to illuminate a light-emitting diode with a coin cell battery (Figure 2g).

### Wicking properties of threads

The wicking property of the plasma-treated cotton threads was characterized by measuring the speed of the water-filling front (Figures 3a and b). The flow followed the standard Washburn relationship in which the filling length is a function of the square root of time29,30,40,41 under controlled environmental humidity (relative humidity ~50%). Evaporation has a significant effect on the wicking properties of the threads41. Evaporation can also affect the concentration of the analytes reaching the sensing zone and may cause errors in the sensing results. However, for implantable devices, where the microfluidic system is embedded within tissue (which has a less differential saturated vapor pressure and comprises a more water-rich environment) evaporation has less of an impact.

### Physical sensors

Physical parameters such as pressure, stress, strain, and temperature are important indicators of tissue health. Here we developed strain and temperature sensors as representative thread-based physical sensors for implantable devices.

To evaluate the performance of the strain sensors, they were stretched at a constant rate (0.1 mm min−1) with a tensile machine (Figure 4e). Strain gauges registered linear changes in electrical resistance when the sensor was stretched. Figures 4f and g show the variation in the relative resistance versus strain for threads coated with carbon ink and CNTs, respectively. The results indicate that the strain sensors made from CNTs were capable of measuring higher strains (up to 100%, GF~3) compared with those made from carbon ink (strains up to 8%, GF~2). This is because CNTs possess higher deformability due to their fibrous structure12. CNTs also offer faster response times and lower creep12.

Another important marker for monitoring tissue health is temperature, as temperature variations can be indicators of inflammation or bacterial infection46. Moreover, temperature measurement is crucial for precise sensing in integrated systems as most chemical and physical biosensors are temperature dependent. Nickel and platinum are the best choices for materials among the readily available metals for resistive temperature sensors. Moreover, they have a linear temperature coefficient of resistance over a long temperature range. However, coating the threads with such materials using inexpensive methods is not feasible. Here we used CNT-coated threads as resistive temperature sensors. Forty centimeters of thread were embroidered in a meandering zigzag pattern on a woven fabric. Such a pattern provided an electrical resistance of 100 Ω in a small footprint. Supplementary Figure S6 shows a linear change in resistance for temperatures ranging from 20 to 40 °C, which is in the biological range of most viable tissues.

### Chemical sensors

pH is one of the most important parameters in the body and is an established indicator of health. Almost all biochemical processes in the body are affected by pH. For example, the pH of a wound is an indication of the wound condition and can be correlated to angiogenesis, protease activity, and bacterial infection47,48. The healing process occurs more readily in an acidic environment; however, extremely acidic pH values can indicate a bacterial infection47. Thus, monitoring the pH of the tissue may provide a method for measuring the condition of the wound bed and ultimately enables determination of the wound’s response to treatment. Another application for pH sensing is in the gastric environment, which is essential for the diagnosis of gastrointestinal diseases, such as inflammatory bowel disease and gastroesophageal reflux disease, or infection from Helicobacter pylori4951.

Here we present a thread-based pH sensor consisting of conductive threads and a microfluidic splitter with three channels for the delivery of a sample to the sensing chambers (Figures 5a and b). The microfluidic splitter was fabricated by patterning hydrophilic threads on a hydrophobic woven fabric. For pH measurements, a potentiometric approach was used. In this method, the open-circuit potential of a working electrode was measured with respect to a reference electrode. CNT coated with doped PANI and silver/silver chloride threads served as the working and reference electrodes, respectively. To evaluate the silver/silver-chloride and doped PANI as electrodes, their conductivity was measured. To perform the experiment, the cotton threads were covered by silver/silver-chloride and doped PANI via several repetitions of dipping and drying. Then, the impedance was measured between two ends. The result is shown in Supplementary Figures S8a, b. Conductivity and charge accumulation on the working electrode are dependent on the protonation and deprotonation of the doped PANI under different pH conditions. The output voltage correlates to the pH value according to the Nernst equation:

$E={E}_{0}-KT/e\left(\mathrm{p}\mathrm{H}\right)$

where E0 is the standard reduction potential of hydrogen with respect to the reference electrode silver/silver chloride, e is the electron charge, and K and T are the Boltzmann constant and the temperature, respectively.

The pH was measured in various buffer solutions with pH values ranging from 3 to 8. All the solutions were prepared using hydrochloric acid and sodium hydroxide in PBS52. This range covers the physiological pH that occurs in the body. Before measurement, each sensor was immersed in purified water to ensure similar starting conditions. The pH of the solution was independently measured using a pH meter (Benchtop pH/MV Meter—860031). A hydrophilic thread was used to deliver the liquid sample from a buffer solution reservoir to the microfluidic thread channel. The thread was passed through a chicken skin to mimic the subcutaneous measurements; the results demonstrate the confinement of biological fluids in the thread-based microfluidic channel without significant leakage onto the chicken fat (Figure 5c and Supplementary Figure S7). Recorded data were sent wirelessly to a desktop computer (Figure 5d). Figure 5e shows the stability of the pH measurement at different acidic/basic conditions. A rapid response time (<30 s) was achieved as the signal was stabilized in a few seconds after changing the pH. The voltage was found to be linearly dependent on the pH value with the slope of −59.63 mV per pH, which exhibits near-ideal Nernstian behavior (Figure 5f). To assess the long-term signal stability, the voltage drop was measured in a solution with a pH of 7.4 for 4 h. A 2.5-mV h−1 drift was observed, indicating the high stability of the fabricated pH sensors (Figure 5g).

The concentration of glucose is an important indicator of diabetes and has to be tightly monitored for the management of this disease. The ability to monitor the glucose level in vivo over a long time would enable the intensive control of blood glucose concentration in patients with diabetes. In addition, such long-lasting implantable sensors reduce the frequency of implantation and replacement, resulting in less discomfort for the patients. Measuring glucose using thread-based sensors is promising for long-term monitoring as threads can be implanted in the body with minimum invasiveness.

Here we developed an amperometric glucose sensor consisting of carbon/functionalized CNT threads (working electrode), carbon threads (counter electrode), and silver/silver chloride threads (reference electrode). Threads were patterned onto a woven fabric as shown in Figure 5h. Glucose oxidase enzyme solution (20 μl, 1 mg ml−1) and 2 ml of nafion (5%) were subsequently added to the working thread. Nafion was used for immobilization of the enzyme53. Glucose solutions with concentrations in the range of 2–15 mM were prepared in PBS with a pH of 7.4. Potassium ferricyanide in KCl (100 mM) was added to the solution as a mediator. Chronoamperometry was used for glucose measurements. Briefly, two pulses with voltages of 0.5 and 0 V with a 50% duty cycle were applied, and the output current was measured. Figure 5i shows the sensor response to different concentrations of the glucose in the solution. Extremely rapid responses (~ms) were observed, with the amplitude depending linearly on the glucose concentration (Figure 5j).

### In vivo evaluation of the integrated thread-based microfluidics, sensors, and electronics

#### Integrated system

A sensing patch using the thread-based toolkit was developed. It consisted of a thread-based microfluidic system with integrated physical and chemical sensors, as described earlier, and a separate readout electronics module. The integration of different components was easily achieved through sewing to form a TDD. The readout electronic circuitry consisted of data acquisition from the sensors, followed by amplification and wireless transmission of the data to a cell phone or computer. A set of off-the-shelf components, including the Texas Instruments LMP91200 and LMP91000 (Texas Instrument, Dallas, TX, USA), was used for measurement of the pH and strain. For measurement of pH-sensing results, the output voltages of the electrodes were attached to an ultralow input current bias buffer amplifier. The output analog voltage was read by an Arduino (SparkFun, Niwot, CO, USA) microcontroller with a 1-Hz sampling frequency. The data were sent to an XBee (SparkFun, Niwot, CO, USA) wireless transmitter. The electronics were implemented by sewing the circuit board on the fabric. Although the board itself was rigid in this initial prototype, it was small and externally placed to not influence the sensor performance. Moreover, one can easily port the electronics onto a flexible printed circuit board on a polyimide substrate through commercial vendors or explore emerging paper/textile substrates5456.

#### In vivo measurements

The gastric and subcutaneous pH values were measured as a representative form of chemical sensing in vivo. For gastric pH measurements, the thread-based sensor was directly inserted into the stomachs of animals (rats) using oral gavage needles as guides (Figure 6a). Subcutaneous measurements were performed by implanting the electrodes under the skin (Figure 6c), or by placing them under the skin using needles (Figure 6d). The results obtained from measuring the pH subcutaneously and in the stomach are shown in Figure 6f. The pH measured under the skin was neutral and stable over 1 min of measurement (pH=7.0±0.1). As expected, the pH value in the stomach was highly acidic and fluctuated from 1 to 3 over time.

The in vivo measurement of the pH was recorded for a short period of time. It demonstrated the feasibility of the thread-based pH sensors as implantable devices. The characterization of the pH sensor in Figure 5g shows the stability and the drift of the sensor over time. The performance of the sensor might degrade when used continuously for an in vivo application due to the adsorption of proteins and other molecules. Antifouling-coating material will be necessary to improve the lifetime of the sensor, but this is not the goal of this study, and an excellent review on the options available can be found elsewhere57.

Suture strain measurements were performed on three wound conditions to mimic the wound closure process during healing: (i) closed; (ii) semi-closed; and (iii) open. For this measurement, a 1-cm incision was made on the back of the neck. The strain sensor was then passed through the wound and secured with a simple knot on each side. Strain-monitoring equipment was then attached using alligator clips. For different wound conditions, the wound site was closed manually, and the strain was measured. The recorded data were then transferred to a mobile phone wirelessly and saved for further analysis. Figure 6h shows the typical values measured by this system for the wound closure model. Note that the strain of the wound was created manually; therefore, the indicated values in the x axes are exact numbers, matching the value taken from the calibration plot of the strain sensor.

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## Acknowledgements

The National Science Foundation partially funded this project under grant EFRI-1240443. SEM images were acquired at Harvard University Center for Nanoscale System. We also acknowledge Sara Mostafalu for her help with embroidering and experiments.

## Author information

Authors

### Corresponding author

Correspondence to Sameer R. Sonkusale.

## Ethics declarations

### Competing interests

The authors declare no conflict of interest.

Supplementary Information for this article can be found on the Microsystems & Nanoengineering website (http://www.nature.com/micronano).

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Mostafalu, P., Akbari, M., Alberti, K. et al. A toolkit of thread-based microfluidics, sensors, and electronics for 3D tissue embedding for medical diagnostics. Microsyst Nanoeng 2, 16039 (2016). https://doi.org/10.1038/micronano.2016.39

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• DOI: https://doi.org/10.1038/micronano.2016.39

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