An Impedimetric Sensing Probe Based on Printed Circuit Board Technology for Monitoring in Cryobiology Applications

Abstract

Cryopreservation of living cells is an effective tool for protection, maintenance, and distribution of genetic resources, which involves exposure to cryogenic temperatures and requires precise control over various parameters to avoid potential cell damages. Hundreds of protocols have been reported for cryopreservation of aquatic species, but replicating them is challenging without a reliable monitoring technique during a cryopreservation process. In this work, we aim to use electrical impedance as a monitoring parameter to assist standardization of cryopreservation processes and reporting. Specifically, this paper reports an impedance sensing probe compatible with cryogenic temperatures and conventional containers in cryopreservation of aquatic species based on printed circuit board technology its characterization in cryopreservation conditions including different sperm extenders (buffer) compositions and concentrations, presence of cryoprotectant, and multiple cooling rates. The developed probe based on printed circuit board (PCB) technology shows a capability of measuring conditions during cryopreservation differentiating among samples with different buffer contents and cryoprotectants. The probe also demonstrates the capability to distinguish different cooling regimes and detect phase change phenomena. This PCB-based sensing platform provides quantitative impedance measurement data during the cryopreservation process at sample preparation, cooling, and while frozen. Technology such as this offers opportunities for improving the reproducibility of protocols generated by the aquatic species community and can be made widely available as open hardware.

Since the 1950 s cryopreservation techniques have been advanced as an efficient tool for conserving genetic resources of agricultural animals and imperiled species.¹ Cryopreservation is a process by which living cells are preserved at ultra-low temperatures, and it allows storage of cells or tissues for extended periods by minimizing metabolic activity.² However, exposure of living cells to cryogenic temperatures can cause detrimental effects to their structure and functionality. Therefore, procedures and conditions play a crucial role in the recovery rate of frozen cells. These can vary drastically depending on the cell type and species, and the skill, experience and goals of the practitioner.

In particular, the cryopreservation of aquatic species germplasm requires significant enhancement.³ For the past 70 years, the problems of cryopreservation of germplasm of aquatic species have been addressed by an almost exclusive focus on development of hundreds of protocols.^4,5 However, most of these protocols suffer from a lack of reproducibility because of variations in sample preparation, freezing, storage, thawing, and most importantly in reporting.⁴ For example, the cooling regime is among the most critical factors in cryopreservation.³ A wide variety of freezing apparatus (from nitrogen vapor inside a polystyrene foam container to high-end programmable freezers) have been used to freeze spermatozoa.⁶ Multiple other factors, such as the cell media and the concentration of cryoprotectant, can also affect the outcomes. The lack of quantitative data in addition to temperature measurements limits the possibility of reproducing protocols and makes comparisons among studies problematic or impossible. The adoption of novel measurement methods would greatly facilitate direct comparisons among implemented cryopreservation protocols and experimental results.

Since introduction of the electrical impedance concept by Oliver Heaviside in the 19th century, it has gained popularity as a powerful tool for many practical applications such as analysis of the electrical properties of bulk materials and biological cell suspensions.^7,8 In our previous work, direct current (DC) resistivity of samples was used to monitor phase transition phenomena during cryopreservation.⁹ To the best of our knowledge, this work represents the only sensing or monitoring system that has been reported to gather such information during cryopreservation procedures inside conventional cryogenic containers such as French straws for research and commercial purposes. To measure resistivity, probes were fabricated by 3-dimensional (3-D) printing and embedding copper wires as the electrodes. Although the 3-D printed materials were compatible with cryogenic temperatures,¹⁰ the manual process of embedding the electrode decreased the consistency of the sensors and control over the device parameters. Also, the measurement of DC resistivity can lead to accumulation of ions at the surface of electrodes, which interferes the accuracy of such measurement. In addition, DC resistivity cannot address the dielectric properties of samples, which has additional importance in solid phase analysis.⁶

The printed circuit board (PCB) has become a fundamental component of most electronic systems. These boards are composed of conducting patterns formed on an insulating substrate made from glass-reinforced epoxy laminate material. The most common material is FR-4 (Flame Retardant) designated by the National Electrical Manufacturers Association (NEMA). Electronic components such as semiconductor chips, capacitors, and resistors can be soldered on a designated footprint on the board, and conducting patterns (traces) are used to link them together. The boards are fabricated in one or multiple layers, and the layers can be connected by use of drilled holes with a conducting inner surface called “vias.”

Sensors and biosensors based on PCB technology have been implemented by several research groups. For example, an electrochemical biosensor array on a PCB substrate was used with polymerase chain reaction (PCR).¹¹ Also, a PCB-based hydrogen peroxide sensor was reported by using gold-plated pads on the PCB.¹² This versatile fabrication method was also adopted to develop sensors for determining the level of nitrate in aqueous solutions.¹³ To the best of our knowledge, PCB platforms have not been reported for impedimetric sensing as a monitoring parameter in cryopreservation procedures.

The goal of this work was to introduce electrical impedance measurement as an effective tool to compare variations induced during implementing cryopreservation protocols. The utilization of impedance as a quality control parameter could also eventually lead to standardization and improvement of the reproducibility of reported cryopreservation protocols. To move toward standardization more people need to adopt such monitoring. Therefore, probes and measurement systems need to be easily accessible to users. To achieve this, open-hardware methodologies can be considered during the design process.¹⁴ Our objectives were to design, fabricate, and characterize a PCB-based impedance sensing probe in conjunction with a portable complex impedance and temperature measurement system for cryopreservation of aquatic species, and to characterize this system in cryopreservation conditions with different buffer compositions and concentrations, presence of cryoprotectant, and multiple cooling rates.

In this work, we developed an impedance sensing probe based on PCB technology to monitor cryopreservation processes. A stand-alone portable complex impedance measurement system was implemented to record the impedance and temperature during the process. The system was able to differentiate samples containing various ionic contents and cryoprotectant. Variations in cryopreservation protocols were distinguished at sample preparation, freezing, and storage stages.

Experimental

PCB-based sensing probe design, fabrication, and characterization.—

In prototyping of instrumentation for cryopreservation applications, multiple constraints needed to be considered such as the cryogenic temperature of liquid nitrogen and dimensions of the common sample containers, in this case 0.5-ml French straws. Printed circuit board technology was used to fabricate pairs of electrodes required for impedance measurement. The sensing probe was designed using a PCB design software (Autodesk Eagle, Version 9.4.0) and was fabricated by an outsource manufacturer (Oshpark LLC, Lake Oswego, OR, US). The fabricated PCBs were based on FR4 substrate (KB6167F, Kingboard Copper Clad Laminate, Shenzhen, China) with a thickness of 0.8 mm. The exposed conductive pads were covered with a thin layer of gold through a process called Electroless Nickel Immersion Gold (ENIG) to avoid oxidation of the underlying copper pads. The sensing probe was composed of three detection zones distributed with equal spacing across a thin stick (Fig. 1A).

Figure 1.

(A) Schematic diagram of the PCB design used for fabrication of the sensing probes (inset: enlarged view of a detection zone). (B) Optical image of an actual fabricated PCB impedance probe with three detection zones and an electrical connector (upper panel). The probe was designed to be positioned within a 0.5-ml French straw, a standard container (lower panel).

The width of the stick was constrained to 2.5 mm to make it possible to fit the probe within 0.5-ml French straws (a standard freezing container). An electrical connector was embedded on the wide end of the probe to facilitate the external electrical connections. Each detection zone was composed of two parallel and planar electrodes with dimensions of 0.2 × 5 mm and a spacing of 0.2 mm (Fig. 1B). Only the electrodes were exposed to the sample solutions. The conductive traces, and the bottom and top layers of the PCB were covered with an insulating material (solder mask) (PSR-4000BN, Taiyo America, Carson City, NV, US). The FR4 PCB substrate was exposed to the solution at the edges. All the conductive features were on the top layer of the PCB to avoid interference from the exposed vias.

All the impedance measurement experiments were carried out using the above-mentioned impedance measurement probe. However, to evaluate the influence of cryogenic temperatures on electrode structure, additional test probes with a larger surface area and smaller width were specifically designed and fabricated (Fig. 2A). These probes had a single electrode with dimensions of 160 × 0.2 mm, and were used only to assess the possible changes in the characteristics of the electrodes on the PCB after exposure to liquid nitrogen. Five test probes were repeatedly placed under liquid nitrogen for 30 s, followed by holding at room temperature for another 30 s. This cycle was repeated 30 times. In addition, to assess the effect of continuous exposure of probes to cryogenic temperature, they were placed under liquid nitrogen for 15 min. The electrical resistance of electrodes was measured before exposure to liquid nitrogen, after 10 cycles, after 30 cycles, and after 15 min of continuous exposure to liquid nitrogen. Also, the electrode surface was evaluated using optical microscopy (Optiphot-2, Nikon, Tokyo, Japan) for any physical damage due to the exposure to ultralow temperatures or significant temperature fluctuations.

Figure 2.

A test probe was specifically designed and fabricated to evaluate the influence of cryogenic temperatures on the PCB electrode structure (A). The surfaces of electrodes were examined by optical microscopy (10-x) before exposure to liquid nitrogen (B), after plunging in liquid nitrogen 10 times (C) or 30 times (D), and after continuous exposure to liquid nitrogen for 15 min (E).

A portable impedance and temperature measurement system.—

The four main components of the measurement system were the control board, impedance measurement circuit, temperature measurement circuit, and user interface. A Raspberry Pi 3 (Model B+) was used as the control unit due to its low cost, small size, customizability and open-source software (Raspberry Pi Foundation, Cambridge, UK). Raspberry Pi is an electronic board that runs Linux-based operating systems with the capability to communicate with multiple peripheral devices.¹⁵ In addition, the network communication compatibility of these boards makes it possible to control device remotely and transfer data to an external server or database.¹⁶

The impedance measurement system was based on the AD5934 chip (Analog Devices, Norwood, MA, US) which is a precision impedance converter system and is capable of communicating with a control board through I²C protocol. The system was capable of stimulating an external complex impedance at known frequencies as high as 120 kHz by utilizing an on-chip programmable direct digital synthesizer (DDS) and an external 16 MHz resonator. The amplitude of the excitation signal could be adjusted to 200, 400, 1000, and 2000 mV with a 3.3 V supply voltage. The response signal was recorded by an on-chip 12-bit analog-to-digital converter (ADC), and it was further processed by a built-in digital signal processor (DSP) to return the imaginary and real part of the measured impedance through a discrete Fourier transform (DFT) algorithm. The measurement range was determined by choosing the proper feedback resistor at the current-to-voltage amplifier of the receiver stage.

Multiple single-throw switches (ADG715, Analog Device, Norwood, MA) with low on-resistance (2.5 Ω) were used to choose between different feedback resistors and on-board calibration impedances. In addition, by using the same switches, the system was also capable of measuring impedance at 16-channels. The switches were controlled by the Raspberry Pi board through I²C protocol.

The gain factor and system phase of the impedance measurement device were calibrated with the help of on-board calibration resistors. The calibration was done at all required frequencies before performing experiments. To characterize the accuracy of the system, external resistive and capacitive loads were employed to assess performance. The resistive load was a 10 kΩ resistor in parallel with a ceramic capacitor with values of 0, 1, and 2 nF.

A high-precision temperature measurement module was integrated to monitor the sample temperature as a factor to be considered in cryopreservation. The temperature measurement unit was based on a MAX31856, precision thermocouple-to-digital converter (Maxim Integrated, San Jose, CA, US). This unit was controlled by the Raspberry Pi through the serial peripheral interface (SPI) protocol. Type T thermocouples (5TC-TT-T-40–36, Omega, Norwalk, CT, US) were used in conjunction with the temperature measurement unit to record sample temperatures in experiments. The thermocouple was placed at the same position as the middle detection zone on the opposite (back) side of the PCB probe to avoid interference with impedance measurements.

Users were able to interact with the system through a 7-inch touch display (Raspberry Pi Foundation, Cambridge, UK). The LCD was connected to the control board by display serial interface (DSI) in addition to I²C protocol connection to detect and utilize touch commands.

Characterization of the PCB probe and the measurement system in cryopreservation conditions.—

The system was characterized by use of different sample compositions and various freezing conditions to assess the capability of impedance measurement to determine the variation in cryopreservation procedures. The cell media used for cryopreservation are typically salt-based buffer solutions containing cryoprotectants. Hanks’ balanced salt solution (HBSS) (306 mOsm kg⁻¹) and 0.9% sodium chloride solution (NaCl) were used for the study because they are widely used in the preparation of fish and shellfish germplasm samples for cryopreservation. Also, to evaluate the capabilities of impedance in determining variation in salt concentration, samples containing 0.45% and 0.22% NaCl were prepared. Samples with 5% and 10% glycerol were also prepared to study the effect of the addition of cryoprotectant on impedance at room temperature. Deionized water was utilized as a control sample. Because cell medium and freezing conditions are major physical factors in replication of cryopreservation protocols, living cells were not incorporated into this feasibility study to avoid additional biological complexity which was beyond the scope of this project.

All sample solutions were loaded within 0.5-ml French straws, and the PCB probe with a Type T thermocouple was placed inside the straw such that the stick was completely inside and the wide end remained outside. The impedance between the electrodes was recorded by the device at room temperature (25 ± 1 °C). Because a programmable freezer with a uniform temperature chamber and horizontal freezing was used, only the middle detection zone was utilized (freezing of straws in a vertical position could have introduced differential temperature zones). The excitation voltage was 200 mV, and the frequency was swept from 2 to 120 kHz at intervals of 500 Hz.

To evaluate the effect of freezing rate and phase-change phenomena on sample impedance, 0.9% NaCl solution was frozen using a programmable freezer at cooling rates of 5 °C, 10 °C, 20 °C, and 30 °C min⁻¹. The chamber temperature was decreased from 25 ° C to −50 °C, while the impedance between the electrodes and temperature of the sample were recorded by the device. In these measurements, the excitation voltage was 200 mV, and the frequency was adjusted to 100 kHz. This frequency was chosen because of possible future applications in the investigation of sperm cell dielectric properties.

In addition, to investigate the dependence of impedance on ice morphology, impedance measurements were made in deionized water and 10% glycerol samples at −50 °C. The impedance measurement was carried out from 2 to 120 kHz at intervals of 500 Hz.

Results and Discussion

PCB electrode.—

The fabricated impedance measurement probe was able to fit securely within a 0.5-ml French straw (Fig. 1B). French straws are conventional containers for livestock semen and have been adopted for use in aquatic species cryopreservation.¹⁷ Printed circuit board technology offers unique properties making it a promising fabrication method for various sensors. Based on the rapid manufacturing advances within this industry, PCBs can be produced inexpensively in large scale due to the increasing number of competitive fabrication services.¹⁸ In addition, use of a PCB-based sensor facilitates integration of the sensor with other electronics.

Exposure of the FR4 substrate to cryogenic temperatures could influence the mechanical properties of the composite drastically.¹⁹ In addition, the difference between the thermal expansion coefficient of the conductive patterns and the substrate imposes additional potential mechanical stress with large temperature fluctuations and can lead to the failure of the material in the form of cracks or mechanical defects. No noticeable cracks or mechanical failures were found on the electrodes after periodic and continuous exposure of the test PCB probes to the liquid nitrogen (Fig. 2). In addition, due to the high length and small intersection area of the electrodes on the test PCB probes, nonobservable cracks or deformities can increase the electrical resistance of electrodes. The electrical resistance values of electrodes were measured before exposure to liquid nitrogen, after plunging in liquid nitrogen periodically, and after continuous exposure to liquid nitrogen (Table I). A repeated-measures ANOVA test was applied to the obtained data, and there was no statistically significant difference in the electrical resistance values before and after exposure to liquid nitrogen periodically and continuously (P-value > 0.05). Therefore, it is likely that PCB probes can be employed to measure impedance in cryogenic applications, which typically are not colder than liquid nitrogen temperatures.

Table I.

The electrical resistance values of printed circuit board electrodes measured before exposure to liquid nitrogen, and after plunging in liquid nitrogen periodically and continuously.

	Electrical Resistance [Ω]
	Before exposure to liquid nitrogen	After plunging in liquid nitrogen 10 times	After plunging in liquid nitrogen 30 times	After exposure to liquid nitrogen for 15 min
Test Probe 1	0.647	0.64	0.653	0.642
Test Probe 2	0.616	0.628	0.627	0.62
Test Probe 3	0.52	0.544	0.538	0.526
Test Probe 4	0.616	0.61	0.613	0.619
Test Probe 5	0.506	0.518	0.511	0.512
Mean Value	0.581	0.588	0.5884	0.5838
Standard Deviation	0.0568	0.0482	0.0544	0.0537

The stable thermal, mechanical, and electronic properties of PCBs have widened their application in developing sensors for different working conditions. More importantly, the inert nature of the materials used in PCB manufacturing minimizes reactions with chemicals used in biomedical and environmental analyses, makes these materials good candidates for use as sensors in a liquid environment.²⁰ In particular, considering the chemical, thermal, and mechanical stability of PCB boards, they provide great potential for use in developing sensors at low cost for extreme sensing conditions such as cryogenic temperatures.

Portable impedance and temperature measurement system.—

The impedance measurement board was fabricated to work a peripheral device communicating with a Raspberry Pi using a two-wire I²C connection (Fig. 3). The device performance was evaluated by measuring externally known impedances. The impedance magnitude (|Z|) and phase (<θ) of an external 10 kΩ resistor in parallel with a ceramic capacitor with values of 0, 1 and 2 nF were measured (Fig. 4). The measured value is in good accordance with the calculated theoretical value. The highest variation between the theoretical and experimental values was during phase measurement of 10 kΩ resistor in parallel with 2 nF capacitor, and the root-mean-square error calculated for this curve was 3.69. The small deviation between the phase values at higher frequencies was due to the additional impedance introduced by the measurement setup (the breadboard and wires) and the tolerance of the components.

Figure 3.

(A) Top-view with labeled components of the 16-channel complex impedance measurement board developed for multiplex measurements and (B) a block diagram of the impedance measurement system.

Figure 4.

The magnitude (A) and phase (B) of the theoretical and measured impedance values obtained using the implemented system and external test load. (10 kΩ resistive load in parallel with a 0, 1, and 2 nF capacitor).

Characterization of the PCB probe and the measurement system in cryopreservation conditions.—

The lack of reproducibility in replicating reported cryopreservation protocols can originate from many sources, including sample processing. Cell media content and concentrations can be a source of variation. Germplasm media (e.g. extender solutions) in cryopreservation are typically composed of a salt solution containing cryoprotectant. Measuring the impedance before and after cell suspension could provide information about the sample content, and could offer a method for validation of cryopreservation protocols through quality assurance.

These salt solutions are commonly isotonic for fish cells to maintain the osmotic balance of the media and intracellular contents to avoid osmotic effects and consequent damage. For most animal cells, 0.9% NaCl is a common isotonic solution. A buffer solution that is widely used to prepare the germplasm samples for aquatic species is HBSS which stabilizes the sample pH and osmotic balance. Cryoprotectants are another crucial additive in the sample preparation process for cryopreservation. The addition of cryoprotectants can limit the probability of injuries to cells during freezing and thawing.²¹ The mechanical damage to cells by: 1) ice crystals and 2) osmotic effects caused by excessive intracellular concentrations of solutes are the main reasons for freezing injuries.²²

Glycerol was chosen in this study because it is an example of a cryoprotectant that penetrates cells and has been utilized extensively to freeze spermatozoa.^23–25 The impedance and temperature were measured and recorded for 10% glycerol, 5% glycerol, deionized water, HBSS (306 mOsm kg⁻¹), and 0.9% NaCl at the frequency range of 2 to 100 kHz (Fig. 5A). Compared to deionized water, the addition of glycerol caused a decrease in the magnitude of the impedance. The resistivity of deionized water at 25 °C is 18.15 × 10⁶ Ω cm⁻¹, and by addition of glycerol with a resistivity of 15.62 × 10⁶ Ω cm⁻¹ impedance decreased.²⁶ In addition, the conductivity of the ionic solutions attributed to the different charge carriers can be calculated based on the molar conductivity of charged particles. NaCl and HBSS samples have different impedance magnitude profiles due to the difference in the conductivity of these solutions (Fig. 5A).

Figure 5.

The magnitude of measured impedance for 0.9% NaCl, HBSS, 10% glycerol, 5% glycerol, and DI water samples (A). The magnitude of measured impedance for 0.22%, 0.45%, 0.90% NaCl (B). The PCB probe and impedance measurement system were used to record at frequencies from 2 to 120 kHz at room temperature (25 ± 1 °C).

With the addition of NaCl the magnitude of impedance decreases. The impedance magnitude was also measured for 0.9%, 0.45%, and 0.22% NaCl at frequencies from 2 to 120 kHz (Fig. 5B). The sample containing 0.9% NaCl showed lower resistivity compared to 0.45% and 0.22% NaCl samples. The increase in ion concentration as charge carriers reduced the sample resistivity and, consequently, the magnitude of impedance between the electrodes of the sensing probe. Therefore, the probe could be used to identify variations in ionic concentration of the buffer solution, which could identify detrimental osmotic effects on the cells. The impedance magnitude was used because solute concentration can have a minor effect on the sample impedance phase.

Considering the different impedance profiles obtained for various sample solutions, the PCB probe and impedance measurements could provide valuable information about commonly used sample contents in cryopreservation. Although conductivity measurements in various samples have been employed previously to estimate ionic strength of solutions²⁷ or total dissolved solids,²⁸ they have not been utilized as part of a monitoring system for cryopreservation applications. Impedance measurements could be used as a quality control factor for the validation of sample preparation in cryopreservation procedures, which cannot be accomplished by temperature measurement alone.

Another important factor in cryopreservation of living cells is the cooling regime in which ice crystals are formed and cell dehydration happens. Cells can endure storage at low temperatures. However, the intermediate temperature zone from −5 °C to −40 °C can be lethal to cells.²² Fast cooling rates minimize the intracellular water loss during freezing and can lead to intracellular ice formation (IFF) due to a high degree of supercooling inside the cells.²⁹ In most cases, intracellular ice formation (IFF) results in damage or death of the cells.³⁰ At slower cooling rates, cells will be exposed to high concentrations of solutes and potentially severe shrinkage due to osmosis.³¹ At desired moderate cooling rates, the intracellular water loss increases the solute concentration inside the cell to survivable levels. This increased concentration of solute suppresses the intracellular fluid freezing point and minimizes the possibility of IFF.³² Therefore, accurate monitoring of the freezing process can ensure desired outcomes from cryopreservation.

Although temperature measurement can provide information about cooling regimes, it is only a function of the thermal conductivity of the sample and is independent from the composition of the sample. The dependence of the impedance on the characteristics of charge carriers and the dielectric properties of samples provides additional information for evaluation of cryopreservation. Impedance was recorded at 100 kHz by the PCB probe for 0.9% NaCl solution undergoing freezing at different cooling rates (Fig. 6). The cooling process affected the impedance by multiple phenomena including temperature profile. The mobility of ions is a strong function of the temperature. The impedance magnitude (Fig. 6A) and phase (Fig. 6B) increased at more rapid rates for faster cooling rates because a decrease in the temperature led to lower conductivity and consequently, higher impedance magnitude.

Figure 6.

The magnitude of measured impedance (A), impedance phase (B), and temperature (C) for 0.9% NaCl as recorded by the PCB probe and impedance measurement system. The samples were cooled from 25 to −50 °C at cooling rates of 5, 10, 20, and 30 °C min⁻¹.

The physical state of the matter also changes during freezing, which can physically impede ion migration. Impedance values were recorded at the phase change zone during the freezing of 0.9% NaCl with a cooling rate of −10 °C min⁻¹ (Fig. 7). The impedance magnitude (Fig. 7A) underwent an abrupt increase after phase change occurred and the latent heat was removed from the system. In the case of an aqueous solution, the impedance is predominantly a function of the number, charge, and mobility of ions.³³ However, after freezing, ice crystals minimize the migration of ions, and electrical current passes predominantly as the result of the transfer of protons.³⁴ This decrease in the number of charge carriers led to an abrupt increase in the magnitude of impedance during ice crystal growth.

Figure 7.

A zoomed-in view of the measured impedance magnitude (A), impedance phase (B), and temperature (red line) at the phase change zone during freezing of 0.9% NaCl with a cooling rate of −10 °C min⁻¹.

Dielectric properties of sample impedance can also change during freezing. Impedance phase component decreased at a higher rate in the cooling regimes with higher freezing rates for 0.9% NaCl (Fig. 6B). As the sample solidified and ionic mobility decreased substantially, samples showed more capacitive behavior rather than resistive behavior. Ion migration was likely the main cause of the resistive behavior of the sample before freezing, and the lag in the impedance phase component was caused by the increased capacitance between the electrodes after immobilization of the ions. In the liquid phase, the impedance phase component showed less dependence on temperature compared to the magnitude of the impedance (Fig. 7). However, an abrupt increase in phase was observable after nucleation occurred. The impedance phase component decreased while the temperature of the sample was static due to the latent heat removal. This suggested that the phase component of the impedance was more dependent on the physical state of the sample rather than the temperature. Therefore, impedance measurements could potentially be utilized to study ice structure without the necessity of sophisticated equipment.

Furthermore, the utility of impedance measurements is not limited to the aqueous phase or intermediate freezing zones. Samples can be investigated after solidification without thawing. Because it is challenging for cells to endure the intermediate temperature zone (between −5 °C to −40 °C) during storage or thawing, a sensing mechanism to monitor the samples in storage temperature could be of interest. The impedance magnitude and phase were measured at a frequency range from 2 to 120 kHz inside DI water and 10% glycerol sample (Fig. 8). Ice containing glycerol likely had a lower impedance magnitude due to the lower electrical resistivity of glycerol.²⁶ Also, the phase component of impedance showed more lag which could be a result of altered ice morphology in the presence of the cryoprotectant. The noise observed in these graphs appears large because the impedance change range is large. Therefore, the resolution of the measurements is comparatively low, which results in noisy data. A range-switching selector would reduce the noise level, but it could induce additional artifacts to the data due to the switching process.

Figure 8.

The measured impedance magnitude (A), impedance phase (B) for 10% glycerol and DI water samples using the PCB probe and the impedance measurement system at −50 °C.

Multiple physical and chemical processes are involved in the values obtained by measuring impedance inside samples which requires detailed study and is beyond the scope of this report. However, solutions commonly used in cryopreservation applications were distinguishable using the results obtained with the PCB probes. Adjusting the sample solution composition of cell media and cryoprotectant is critical in cryopreservation outcomes. Therefore, numerous cryopreservation protocols have been reported. However, these protocols are difficult to replicate, and temperature profiles are often the only quantitative data to validate the implemented method. Indeed, most studies only include target rates without data³ further demonstrating the need for inexpensive and standardized probes. Compared to impedance measurements, temperature data alone do not provide any information about sample content, and it provides no or only limited information about the nucleation process. Impedance data could also be potentially used to evaluate samples during frozen storage.⁹ If published reports provided temperature and impedance data, this would provide a quantitative means to directly compare protocols, and would provide a new methodology for quality control in repositories. Therefore, the probes could be used to identify variation in cryopreservation protocols in liquid and solid phases as well as during freezing. Thus, the adoption of impedance measurement could provide a simple and economical method to investigate the freezing process and characteristics of frozen samples, for example, for quality control purposes.

In addition, combination of the sensing platform and portable measurement system with wireless data transfer facilitates standardization and data management for small-scale research efforts or large-scale repository development programs, particularly in aquatic species for which reproducibility and standardization are fundamental challenges.⁴ This system could also be used in conjunction with low-cost freezing devices (e.g., fabricated using 3-dimensional printing) to eliminate the necessity of expensive equipment for performing cryopreservation.³⁵ This work will enable research community to compare implemented cryopreservation protocols with original reported protocols. Also, germplasm repositories could validate the freezing process before accepting samples through use of impedance data as a quality assurance parameter to avoid expenses on storage of low-quality samples.

The term “open hardware” refers to devices with publicly available design files associated with certain user agreements and freedoms.¹⁴ Such devices can typically be studied, fabricated, modified, and distributed by interested users. The utilization of this concept in developing instrumentation for research efforts can drastically reduce the cost of scientific hardware for user communities.³⁶ Particularly for the proposed system, the designs of the PCB sensors can be distributed in the form of widespread and customizable files (e.g., Gerber). The sensors can then be replicated by widely available PCB fabrication services. Also, the designed electronic instrumentation utilizes an open-source platform as the control unit (Raspberry Pi). The electronic system design is simple and can be shared in the form of open-source software files (Eagle, Autodesk, San Rafael, CA). The electronic devices can be replicated using the design files and off-the-shelf parts. Also, more skilled users can modify the design based on their needs. As a larger portion of the aquatic species community begins to use such devices, the outcome can be studied to compare or validate cryopreservation, which can improve reproducibility in reported protocols. Besides, the inherent simplicity and low cost of open hardware can facilitate the adoption of cryopreservation by new user groups who were not previously employing cryopreservation due to the complexity or high price of the required equipment.

The work presented herein demonstrates the potential for sensing platforms to facilitate harmonization and standardization in cryopreservation protocols. In addition to impedance, various other sensing mechanisms can be incorporated into a PCB based on user needs. For example, electrochemical^37,38 or optical¹² methods can be utilized to determine the concentration of different target analytes as biomarkers for healthy cell activities.

Conclusions

In summary, we have introduced impedance measurement as an alternative approach for much-needed monitoring of cryopreservation procedures in aquatic species. Printed circuit board technology was utilized to develop impedimetric sensing probes to work at cryogenic temperatures. Such sensors can be manufactured at relatively high scalability and low cost. The design of such sensors can be readily customized based on user needs. Also, a portable complex impedance measurement system was designed and fabricated to record data from the sensors in relation to user information for further processing. Impedance measurements were able to identify variations in sample preparation, freezing, and storage during cryopreservation.

Considering the network compatibility of such systems, the data obtained can be used to improve reproducibility and eventually standardize repository accessions for germplasm and genetic resources of aquatic species. Our approach can potentially improve the validation of cryopreservation protocols when a specific protocol is being replicated, which is one of the critical issues in the field of cryopreservation. In addition, the system can be used to increase the reproducibility of results reported by researchers. The low-cost of the monitoring system reported in this work can encourage more user groups to utilize cryopreservation as an effective tool for preservation of genetic resources. If desired, the resolution of the system could be improved for high-fidelity data acquisition, such as for detailed study of cryobiology. But, at present, it is designed as a much-needed method to provide reproducibility in development, reporting, and use of cryopreservation procedures. Finally, because it was developed as open hardware, we also hope it will provide a baseline for future innovation and community interaction in developing novel sensing and monitoring systems such as these.