MetalâOrganic Framework Encapsulation for Biospecimen

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Metal-Organic Framework Encapsulation for Biospecimen Preservation Congzhou Wang, Hongcheng Sun, Jingyi Luan, Qisheng Jiang, Sirimuvva Tadepalli, Jeremiah J. Morrissey, Evan D. Kharasch, and Srikanth Singamaneni Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b04713 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Chemistry of Materials

Metal-Organic Framework Encapsulation for Biospecimen Preservation Congzhou Wang1, Hongcheng Sun1, Jingyi Luan1, Qisheng Jiang1, Sirimuvva Tadepalli1, Jeremiah J. Morrissey2,3, Evan D. Kharasch2,3,4,5, and Srikanth Singamaneni1,3* 1

Department of Mechanical Engineering and Materials Science, Institute of Materials Science and Engineering, Washington University in St. Louis, Saint Louis, MO 63130, USA

2

Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, 63110, USA

3

Siteman Cancer Center, Washington University in St. Louis, St. Louis, MO, 63110, USA

4

Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO, 63110, USA.

5

The Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University School of Medicine, St. Louis, MO, USA

*To whom correspondence should be addressed: [email protected]

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Abstract: Handling, transport and storage of biospecimens such as blood and urine without refrigeration are extremely challenging.

This formidable challenge leads to an inevitable

reliance on a “cold chain” for shipping, handling and storage of biospecimens throughout the world.

The cold chain requirement precludes biospecimen procurement from underserved

populations and resource-limited settings where refrigeration and electricity are not reliable or even available.

Here, we introduce a universal biospecimen preservation approach based on

nanoporous material encapsulation for preserving protein biomarkers in biofluids under normal (non-refrigerated) storage conditions.

Using urinary NGAL and serum/plasma CA-125 as the

model protein biomarkers, we demonstrate that zeolitic imidazolate framework-8 (ZIF-8), a nanoporous material, encapsulation can preserve protein biomarkers in urine, serum, plasma and blood at room temperature and 40°C, with comparable preservation efficacy to the refrigeration method (freezing liquid samples at -20°C). The protein biomarkers in the relevant biofluids are first encapsulated within the nanoporous crystals (i.e., ZIF-8) and then dried on paper substrates via a dry spot sample collection method, greatly improving biospecimen collection and handling capability in resource-limited settings. Overall, this energy-efficient and environmentally-friendly approach will not only alleviate huge financial and environmental burden associated with “cold chain” facilities but also extend biomedical research benefits to underserved populations by acquiring reliable clinical samples from regions/populations currently inaccessible.


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Introduction The availability of high-quality biospecimens such as blood and urine is critical to biomedical research and clinical diagnosis.1-3

Unfortunately, due to the poor stability of molecular

biomarkers (especially proteins) in these biofluids at ambient and elevated temperatures, they are prone to lose their structure and biofunctionality during the pre-analytical stage (i.e. the preparation, storage or transportation period between sample collection and analysis).4-6 For instance, when refrigeration is not maintained, blood-derived biospecimens degrade quickly, accounting for up to 67% of all laboratory testing errors.5, 7-8 This requires investigators and clinicians to dedicate a significant fraction of budget to refrigeration or “cold chain” costs associated with biospecimen storage or transportation.9-10

More importantly, the refrigeration

and “cold chain” are simply not feasible in resource-limited settings (e.g., low and middle income countries), which hinders biospecimen procurement, disease screening, early diagnosis and therapeutic intervention in underserved populations since it is impractical, if not impossible, to expect patients scattered in rural areas to travel and access an extremely limited number of hospitals and clinical laboratories for screening and diagnosis.11

A feasible solution to the

aforementioned challenges is a refrigeration-free biospecimen preservation technology that can be implemented in resource-limited settings (i.e., absence of electricity, refrigeration, trained personnel) and underserved populations to efficiently collect, process and ship well-preserved biospecimens to centralized clinical/research laboratories and hospitals with qualified facilities for analysis. Considering that the Clinical and Laboratory Standards Institute (CLSI) recommends urinalysis and culture and testing within 2 hours of urine sample collection, urinary samples are often treated with preservatives such as boric acid, tartaric acid, and chlorhexidine to extend the sample storage time if analysis within two hours of sample collection is not feasible.12-13 However, these preservatives are applicable only for short preservation times ranging from 24-72 hours, which are not sufficient for implementation in resource-limited settings where the 3 ACS Paragon Plus Environment


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time lapse between sample collection and analysis can be much longer.

Moreover, the

preservation timescales with the existing preservatives are also not sufficient for research biospecimens used for biomarker discovery and validation, which often involves months to years. Other approaches to dry blood samples, such as isothermal vitrification,14 lyophilization15 or within a solid matrix such as silk fibroin, have been reported.16 In the isothermal vitrification and lyophilization approaches, sugars such as sucrose and trehalose are often added into protein solution in order to form sugar-glass matrix for stabilizing proteins.17 However, vitrification and lyophilization of protein with sugars are difficult to implement in resource-limited settings and in some cases lack of control of sugar crystallization can lead to protein aggregation.18 Drying blood in a silk fibroin matrix represents a novel approach for long-term biofluid preservation without refrigeration. However, the technology involves the use of silk fibroin solution with limited temperature and environmental stability and relatively short shelf-life, making it difficult to implement in resource-limited settings. It is also reported that the addition of zinc ions (such as zinc sulfate) can enhance the stability of specific types of proteins.19-20

Overall, none of the

existing methods can be considered a “universal biospecimen preservation technique”, which would allow both long-term storage without refrigeration and the implementation in resource-limited settings. Metal-organic frameworks (MOFs) are an emerging class of nanoporous materials that are comprised of metal ions or clusters linked by organic ligands.21-22

MOFs are considered to be

highly attractive for a variety of applications, including, gas and energy storage,23-24 drug delivery,25-27 catalysis,28-29 separation,30-31 and chemical sensors.32 Their attractive properties include nanoporous structure with a large surface area, tunable porosity, rich organic functionality, stable shelf-life of precursor materials and excellent thermal stability.33-36 Within the emerging applications, of particular interest is the biopreservation ability of the MOFs, which is believed to rival conventional porous solids and biomaterials.37-38 When incorporated into these nanoporous materials to form MOF biocomposites, the protein molecules will be confined within the rigid framework structures, thus maintaining their structures and activities against 4 ACS Paragon Plus Environment

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denaturation and degradation conditions.39-40 Pioneering work by Falcaro and Tsung’s groups involved spontaneous biomineralization process or a de novo approach, to encapsulate enzymes within MOFs for biocatalysis applications.37, 41-42 We recently demonstrated that MOF (zeolitic imidazolate framework-8, ZIF-8) can be used as a protective material to preserve the recognition capability of antibodies or viral protein on biosensor surfaces stored at ambient and elevated temperatures.43-45 The preserved antibody-based biosensor can be restored at a later time (before use for detection) by dissociating the ZIF-8 layer in an aqueous solution at pH 6. Considering the versatile molecular encapsulation capability of ZIF-8 in aqueous solution, high thermal stability, and ability to be disintegrated upon demand by lowering the pH, we hypothesize that ZIF-8 can serve as a protective encapsulant for preserving the structure of various protein biomarkers in biofluids under unregulated temperatures. In this study, we introduce a novel technique for preserving protein biomarkers in biofluids (urine, serum, plasma and blood), in dry state, by combining MOF-based preservation with dry spot sample collection. The technology introduced here involves simple mixing of biofluid samples with ZIF-8 precursors and drop-casting of a specific volume of the mixture onto a paper substrate to allow air-drying.

This simple and easily deployable process results in encapsulation of

protein biomarkers within ZIF-8 crystals that could be easily dissociated upon demand at a later time. The technique overcomes the drawbacks of conventional dry spot technology (i.e. limited stability of the dried specimen, the need for refrigeration and inaccuracy in determining the concentration of the biomarker) while retaining transportation and storage convenience (Figure 1). We choose neutrophil gelatinase-associated lipocalin (NGAL), a urinary biomarker for acute kidney injury,46-47 and CA-125, a serum/plasma biomarker for ovarian cancer48-49 as the model proteins.

Using the commonly employed bioanalytical tools such as enzyme-linked

immunosorbent assay (ELISA), circular dichroism spectroscopy and fluorescent protein microarray, we demonstrate that the ZIF-8 encapsulation confers excellent structural stability to protein biomarkers in biospecimen in dry state, even when stored at nominal room temperature or high ambient temperatures encountered in different parts of the world. Such a biospecimen 5 ACS Paragon Plus Environment


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technology will not only alleviate huge financial and environmental burdens associated with “cold chain” but also extend biomedical research benefits to underserved populations by acquiring clinical samples from regions/populations currently inaccessible. Results and Discussion As a proof-of-concept, NGAL-spiked artificial urine was employed as a model biospecimen. To prepare a typical ZIF-8 preserved sample, NGAL-spiked artificial urine (50 µg/ml, 25 µl) was first mixed with 2-methylimidazole solution (640 mM, 12.5 µl) and then zinc acetate solution (160 mM, 12.5 µl), and incubated at room temperature for 1 hour.

Subsequently, the mixture (50 µl) was

air-dried on a 0.5×2 cm Whatman 903 paper strip (which usually takes 2 hours at room temperature) (Figure 2A).

Scanning electron microscope (SEM) images show the distinct

morphologies of paper substrates after drying NGAL-spiked artificial urine with and without ZIF-8 precursors (Figure 2B and 2C). The granular morphology of the paper substrate with artificial urine mixed with ZIF-8 precursors suggests the formation of ZIF-8 crystals (Figure 2B). Conversely, the morphology of the sample without adding ZIF-8 precursors (Figure 2C) is similar to bare paper (Figure S1) since the proteins are too small to be visible at this magnification. Owing to their rich functionality (such as carboxyl, carbonyl, hydroxyl, and imidazole groups), proteins serve as nucleation sites for the formation of ZIF-8 crystals.37 Here, we expected that ZIF-8 crystals would form and encapsulate NGAL in the presence of ZIF-8 precursors in the NGAL-spiked artificial urine.

Indeed, the NGAL-embedded ZIF-8 crystals formed after 1 hour

incubation of the mixture solution and could be collected by centrifugation. The SEM and transmission electron microscopy (TEM) images show that the crystals formed in the NGAL-spiked artificial urine exhibit uniform size of ~0.5 µm (Figure 2D), with a rhombic dodecahedral shape, similar to that of pure ZIF-8 crystals (Figure S2).

The powder X-ray

diffraction (XRD) pattern of the crystals also exhibits the typical peaks of pure ZIF-8 crystals (Figure 2E).


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To confirm the encapsulation of NGAL into ZIF-8 crystals, both pure ZIF-8 and NGAL-embedded ZIF-8 crystals were subjected to calcination (325 °C for 2 h). Only NGAL-embedded ZIF-8 showed pores in the calcinated crystals, indicating the encapsulation of protein in the ZIF-8 crystals (Figure S3). To further ascertain the formation of NGAL-embedded ZIF-8 crystals, Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy were employed.

The

FTIR spectrum obtained from the crystals not only shows typical ZIF-8 absorption peaks at 1584 cm-1 (C=N stretching of imidazole) and 1400-1500 cm-1 (the imidazole ring stretching), but also exhibits absorption peaks at 1640-1670 and 1520-1560 cm-1, corresponding to amide I and amide II bands of protein, respectively (Figure 2F).50 In contrast, pure ZIF-8 crystals and NGAL only show their respective characteristic peaks.

Similar results are observed by Raman

spectroscopy, which also indicates the encapsulation of NGAL by ZIF-8 crystals (Figure 2G). The Raman spectrum obtained from pure NGAL exhibits a broad band at 1630-1690 cm-1, corresponding to amide I band of protein.51 The NGAL-encapsulating ZIF-8 crystals exhibit the amide I band of NGAL (not present in pure ZIF-8 crystals) and 2-methylimidazole characteristic bands at 1146, 1182, 1384, 1460 and 1505 cm-1 corresponding to C−N stretching, C−N stretching plus N−H wagging, CH3 bending, C−H wagging and C−N stretching plus N-H wagging, respectively.52

To quantify the encapsulation efficiency, the supernatant after crystals

centrifugation was collected and remaining NGAL concentration was determined using sandwich enzyme-linked immunosorbent assay (ELISA). The encapsulation efficiency was found to be dependent on the concentration of ZIF-8 precursors. Specifically, when the concentrations of zinc acetate and 2-methylimidazole in the mixture increased to 40 mM and 160 mM, respectively, ~95% NGAL was encapsulated within ZIF-8 crystals (Figure S4).

As a control experiment,

simple mixing of NGAL-spiked artificial urine with pure ZIF-8 crystals resulted in extremely low (~10%, owing to the physical adsorption) encapsulation efficiency. This physical mixing of pre-formed ZIF-8 crystals with the protein biomarkers is in stark contrast with the protein-embedding approach (i.e. formation of ZIF-8 crystals in the presence of protein biomarkers), which exhibited high encapsulation efficiency (95%). 7 ACS Paragon Plus Environment


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Next, we set out to evaluate the efficacy of ZIF-8 encapsulation in preserving NGAL upon exposure to harsh conditions (such as elevated temperatures) that would normally lead to protein denaturation and loss of biospecimen integrity.

NGAL-spiked artificial urine dried on

paper substrates with and without ZIF-8 encapsulation were stored at 25, 40, or 60°C for 1 week. Before analysis, the optimized elution buffer (0.2 M phosphate buffer at pH 5.6+Tween 20+EDTA, see Figure S5 and S6 for details) was used to elute NGAL from the paper substrates. The slightly acidic environment (at or below pH 6) was employed to dissociate ZIF-8 crystals and release encapsulated NGAL via breaking coordination between the zinc ions and imidazolate.27 We found that the ZIF-8 encapsulated NGAL dried on the paper was almost fully eluted (˃95%) whereas NGAL alone dried on the paper was only partially eluted (~75%) (Figure S6). It is important to note that ZIF-8 encapsulation prevented irreversible adsorption of proteins on the cellulose fibers by creating a crystal interface between the protein and paper substrate. We also confirmed that the slightly acidic elution buffer and the ZIF-8 residue did not alter the protein characteristics and downstream bioanalysis (Figure S7).

After storage and elution, the

concentration of NGAL in the eluate was quantified using the NGAL sandwich ELISA. The preservation efficacy (preservation %) was calculated by comparing the NGAL amount in the eluate to the spiked NGAL amount in the artificial urine.

As shown in Figure 3A, there was more

than 85% preservation of NGAL with ZIF-8 encapsulation after 1 week storage at 25 and 40°C, as well as more than 80% at 60°C. Notably, at 25 and 40°C, NGAL with ZIF-8 encapsulation showed comparable preservation to freeze-thawed liquid samples (the refrigeration approach as the control) stored at -20 °C. On the other hand, NGAL without ZIF-8 encapsulation stored at these temperatures for 1 week exhibited less than 30% preservation although 70% of the proteins were eluted as measured by bicinchoninic acid (BCA) assay, indicating the denaturation of NGAL under these storage conditions (Figure S8).

To further confirm that ZIF-8

encapsulation preserves the encapsulated protein structure, circular dichroism (CD) spectroscopy was employed to characterize the secondary structure of human serum albumin (HSA) with and without ZIF-8 encapsulation after 1 week incubation at 40 °C (Figure S9). As 8 ACS Paragon Plus Environment

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expected, elevated temperature caused a significant change (a decrease in the α-helical content) of the secondary structure of unencapsulated HSA, as shown in the CD spectrum. In contrast, the secondary structure of ZIF-8 encapsulated HSA was found to be very similar to that of the pristine HSA, indicating that ZIF-8 encapsulation is able to preserve the structure of encapsulated protein. We also found that the preservation efficacy critically depended on the concentrations of 2-methylimidazole and zinc acetate (Figure S10). A low preservation was noted upon using 40 mM 2-methylimidazole with 10 mM zinc acetate, which can be attributed to incomplete encapsulation of NGAL under this condition (~70% encapsulation of NGAL using 40 mM 2-methylimidazole with 10 mM zinc acetate, Figure S4). Subsequently, using the optimal ZIF-8 precursor concentration (160 mM 2-methylimidazole and 40 mM zinc acetate), we extended the storage time under different temperatures up to 4 weeks (Figure 3B). Different dried paper strips were sampled at selected time points (2, 3 or 4 weeks) to monitor possible changes in NGAL preservation. With ZIF-8 encapsulation, over 70% of NGAL on paper was preserved up to 4 weeks (the maximum time tested) at all three temperatures (25, 40 and 60°C), as opposed to 20% preservation on samples without ZIF-8 encapsulation.

Notably, the ZIF-8 encapsulation

had comparable preservation (~90%, at 25 and 40°C) to the freezing method over 2 weeks (-20°C).

Remarkably, 70% of NGAL on paper could be preserved within 4 weeks at 60 °C,

which represented an extremely harsh storage condition (a surrogate for long-term storage stability at room temperature). Following the successful optimization of ZIF-8 encapsulation using spiked artificial urine samples, we set out to evaluate the applicability of this technique to patient urine samples. Three acute kidney injury patients with different urinary NGAL levels (Patient #1: 32.8 ng/ml, Patient #2: 17.4 ng/ml and Patient #3: 97.5 ng/ml, quantified by ELISA and confirmed by Western blotting, Figure S11) were selected.

Compared to artificial urine, human urine is more

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even whole cells (e.g., red blood cells and shed kidney cells).53 As routinely done, the cells were removed by low speed centrifugation before ZIF-8 encapsulation. We confirmed that mixing patient urine with ZIF-8 precursors resulted in a solution at near neutral pH. Thus, protein biomarkers in the mixture are not subjected to extremely low or high pH (Table S1). To assess the preservation efficacy of NGAL, the urine samples with and without ZIF-8 encapsulation dried on paper substrates were stored at 25 or 40°C for 1 week. concentration in the eluate was quantified using ELISA.

NGAL

For all three patients, the samples with

ZIF-8 encapsulation resulted in more than 85% NGAL preservation after 1 week storage at both 25 and 40°C, whereas the control samples without ZIF-8 encapsulation showed less than 40% NGAL preservation (Figure 3C). To further demonstrate the capability of this technique to simultaneously preserve multiple biomarkers in patient samples, the recovered urine samples (with and without ZIF-8 encapsulation, after storage at 40°C for 1 week) from Patient #3 were assayed by a multiplexed analysis tool, a protein microarray (Figure 3D and 3E).

The

frozen-liquid samples stored at -20°C were employed as the reference. The results showed that four detectable acute kidney injury biomarkers54-55 (albumin, NGAL, cystatin C and Beta-2 microglobulin) were almost fully (>95%) preserved with ZIF-8 encapsulation, whereas samples without ZIF-8 encapsulation exhibited ~40-60% fluorescence signal intensity loss corresponding to these biomarkers.

Overall, these results clearly demonstrate the efficacy of ZIF-8

encapsulation in preserving the dried urinary protein biomarkers on paper substrates at high temperatures. Finally, we turned our attention to blood (and components serum and plasma), the most common biospecimens in biological and clinical studies.

Compared to urine, serum or plasma

represents a more complex biological matrix due to the presence of large amount of various proteins such as albumin, globulins and fibrinogen.

Before proceeding to assess the

preservation of specific protein biomarker, we confirmed that different types of proteins can be extracted from the paper substrates containing dried serum with or without ZIF-8 encapsulation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating the 10 ACS Paragon Plus Environment

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potential of this technique in preserving multiple protein biomarkers simultaneously in serum or plasma (Figure S12).

CA-125, a serum biomarker for ovarian cancer,48 was used as the model

protein and spiked into serum from healthy people.

Considering extremely high concentration

of serum proteins, the serum was first diluted (5-, 10- or 20-fold) before spiking CA-125 and adding ZIF-8 precursors to ensure ZIF-8 formation and more complete encapsulation. The CA-125 spiked serum samples with and without ZIF-8 encapsulation were dried on paper and stored at 40°C for 1 week. The results indicated that CA-125 spiked in 20-fold diluted serum afforded the highest preservation (~90%, Figure 4A). The poor preservation of CA-125 from 5and 10-fold diluted serum was due to the incomplete ZIF-8 formation and encapsulation (Figure S13). This was further confirmed by XRD and SEM imaging (Figure 4B and 4C), revealing decreased ZIF-8 crystal formation with increased total protein concentration in serum. Subsequently, CA-125 was spiked into three different matrices that represent typical blood-derived biospecimens (serum, heparin-anticoagulated plasma and EDTA-anticoagulated plasma from healthy people with 20-fold dilution), dried on paper substrates with and without ZIF-8 encapsulation, and subsequently stored at 25, 40, or 60°C for different durations of time. CA-125 concentration in the eluate was quantified using sandwich ELISA. In all three matrices, CA-125 with ZIF-8 encapsulation exhibited ~85% preservation after 4 weeks storage at 25 and 40°C, as well as ~75% at 60°C (Figure 4D-F).

On the other hand, CA-125 without ZIF-8

encapsulation stored at these three temperatures for 4 weeks displayed ~50%, 40% and 30% preservation, respectively.

Remarkably, the preservation efficacy of ZIF-8 encapsulated

CA-125 over 4 weeks of storage at 25 and 40°C was comparable to the refrigeration method (freezing liquid samples at -20 °C) for the same storage duration. In the end, we set out to assess the applicability of this technique to preserve blood samples drawn in resource-limited settings.

Unlike serum and plasma, the presence of a large quantity

of whole cells (red and white blood cells and platelets) in blood may affect ZIF-8 encapsulation of target protein biomarkers. Considering that ZIF-8 can also form a thick layer on cell surfaces56 and lead to incomplete encapsulation or preservation of target protein biomarkers, it is important 11 ACS Paragon Plus Environment


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to remove blood cells through centrifugation before ZIF-8 encapsulation of protein biomarkers. Unfortunately, centrifuge is typically inaccessible in resource-limited settings considering that conventional systems are bulky, expensive and electrically-powered.

Here, we implemented a

hand-powered centrifuge introduced by Prakash’s group57 that was able to separate plasma from whole blood within 10 minutes for subsequent ZIF-8 encapsulation (Figure 5A). In order to mimic fresh whole blood samples from ovarian cancer patients, four different concentrations of CA-125 within pathological-relevant range from 25 ng/ml-25 µg/ml were spiked into fresh blood from healthy volunteers.58-59

Then the plasma samples (heparin-anticoagulated) were

separated from blood and diluted 20-fold for ZIF-8 encapsulation. To assess the preservation efficacy of this approach under unregulated conditions, two sets of experiments were devised. First, the ZIF-8 preserved plasma samples with the four different CA-125 concentrations were subjected to temperature fluctuations for 8 days (two days 25°C followed by two days 40 °C as one cycle and run for two cycles, Figure 5B). Second, the ZIF-8 preserved plasma samples with the four different CA-125 concentrations were shipped to California, USA and sent back to Missouri, USA via a regular shipping package (10 days in unknown shipping and handling conditions, Figure 5C).

Unencapsulated samples dried on papers were used as negative

controls in both cases. As shown in Figure 5B and 5C, samples with ZIF-8 encapsulation can achieve up to 90% of preservation, as opposed to ~50-60% of preservation from control samples. It is also important to note that the preservation efficacy did not significantly change with the variation of CA-125 concentrations. Overall, the experiments here clearly demonstrate the feasibility and robustness of this approach in preserving protein biomarkers in blood samples.

By combining with the hand-powered centrifuge, it is possible to directly collect and

preserve fresh blood samples in resource-limited settings. Conclusion In summary, we report a novel biospecimen preservation approach involving the use of a nanoporous material (ZIF-8) as an encapsulant for preserving protein biomarkers in biofluids 12 ACS Paragon Plus Environment

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under non-refrigerated storage conditions. Using urinary NGAL and serum/plasma CA-125 as the model protein biomarkers, we demonstrate that the ZIF-8 encapsulation approach can preserve protein biomarkers in urine, serum, plasma and blood at room temperature and 40°C, with comparable or better preservation efficacy than the refrigeration method (freezing liquid samples at -20°C). The protein can be recovered by dissociating the ZIF-8 protective layer in pH 6 buffer without affecting the protein structural integrity and downstream analysis.

By

combining the MOF-based preservation approach with dry spot sample collection method, the protein biomarker in biofluid can be preserved in dry state, greatly improving the biofluid-related biospecimen collection and handling capability in resource-limited settings.

Overall, this

energy-efficient and environmentally-friendly approach not only represents a novel technique to eliminate the “cold chain” and temperature-controlled handling of biofluid-related biospecimens, but also allows interruptible, storable, and restorable on-demand analysis at a later time in a centralized or distributed location/manner to improve the reliability of the bioanalytical results. We believe that this facile and low-cost approach would open up the new avenues in both research and clinical settings such as large-scale cancer screening, epidemiologic studies from tropical and disaster-struck areas, remote chronic disease monitoring and clinical trials for new drugs.

We envision a “Biopreservation Kit” containing ZIF-8 precursors, paper strips and

transfer pipettes, enabling patients to self-prepare dried blood/urine samples and send to hospitals or clinical labs via regular mailing.

Ultimately, this approach can alleviate hospital and

logistical burdens, facilitate disease monitoring and patient feedback, and offer new services for currently underserved populations. Experimental section Chemicals.

2-methylimidazole, zinc acetate dihydrate, ethylenediaminetetraacetic acid

(EDTA), Tween 20, sodium phosphate monobasic and sodium phosphate dibasic were purchased from Sigma-Aldrich. Artificial urine (Surine™ Negative Urine Control) was purchased from Cerilliant Company (a Sigma-Aldrich Company). This artificial urine is suitable for LC/MS or 13 ACS Paragon Plus Environment


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GC/MS applications in clinical chemistry, urine drug testing or forensic analysis. No protein and preservatives are added.

Human recombinant neutrophil gelatinase-associated lipocalin

(NGAL), human recombinant CA-125, NGAL ELISA kit (DY1757, detection range: 78 pg/ml to 5000 pg/ml), and CA-125 ELISA kit (DY5609, detection range: 31.2 pg/ml to 2000 pg/ml) were purchased from R&D systems. Custom protein microarray kits were purchased from RayBiotech (Custom G-Series Antibody Array, AAX-CUST-G).

Pierce bicinchoninic acid (BCA) protein

assay kit was obtained from Thermo Fisher Scientific.

For patient samples, approval was

obtained from the Washington University Institutional Review Board, and written informed consent was obtained from all patients. Sample preparation. NGAL-spiked artificial urine (50 µg/ml of NGAL, 25 µl) or patient urine (25 µl) was first mixed with 2-methylimidazole aqueous solution (12.5 µl) and then zinc acetate dihydrate aqueous solution (12.5 µl). The final concentrations of 2-methylimidazole after mixing were 320 mM, 160 mM, 80 mM and 40 mM. The final concentrations of zinc acetate dihydrate after mixing were 80 mM, 40 mM, 20 mM and 10 mM.

Note that the molar ratio of

2-methylimidazole to zinc acetate dihydrate were kept at 4:1. After 1 hour incubation at room temperature (20-23 °C), 50 µl mixture was transferred onto a 2x0.5 cm Whatman 903 paper strip (Sigma) to allow air-drying (usually about 2 hours at room temperature).

For accurately

determining the NGAL recovery after storage, it is important to avoid liquid leakage from the paper strip during the drop-cast process. Typically, a 2×0.5 cm paper strip can absorb 50 µl biofluid without leakage. After drying, the paper strips were sealed in petri dishes and stored at 25 °C, 40 °C or 60 °C for different time intervals. The sample preparation for serum and plasma were similar to urine except that the serum and plasma were first diluted 5, 10 or 20 times with PBS and then spiked with CA-125 (50 µg/ml of CA-125). For the fresh blood samples, the CA-125 was first spiked into the blood and then the plasma was separated from the blood using hand-powered centrifuge for the subsequent sample preparation.


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Protein recovery. Before analysis, the paper strips were eluted in 1 ml elution buffer (0.2 M phosphate buffer with 2 mM EDTA and 0.1% Tween 20 at pH=5.6) by shaking the paper strip in a cuvette with the elution buffer at the speed of 60 r. p. m. for 1 hour. Different elution buffer recipes were tested for maximal recovery (Figure S5 and S6). The elution solution was then assayed by ELISA for the target analyte. NGAL and CA-125 standards provided with the ELISA kits were used to generate a standard curve for each assay. Characterization. To characterize protein-embedded crystals, crystals were centrifuged and washed with DI water twice (8k r. p. m. for 20 minutes).

To calculate the encapsulation

efficiency, the supernatant after first centrifugation was collected and assayed by ELISA. SEM images were obtained using a FEI Nova 2300 field-emission scanning electron microscope at an acceleration voltage of 10 kV. Fourier transform infrared spectroscopy (FTIR) measurements were conducted using a Nicolette Nexus 470 spectrometer. The Raman spectra were obtained using a Renishaw inVia confocal Raman spectrometer mounted on a Leica microscope with a 50× objective and a 514 nm wavelength diode laser as an illumination source. The X-ray diffraction (XRD) measurements of the samples were recorded on a Bruker D8-Advance X-ray powder diffractometer using Cu Kα radiation (λ = 1.5406 Å). SDS-PAGE and Western blotting protocols. For SDS-PAGE, 300 µl of eluate was mixed with 0.9 ml acetone-methanol (1:1), placed on ice for 1 hour and centrifuged at 10,000 r. p. m. for 10 minutes to precipitate and concentrate the eluted urine or serum proteins. The protein pellet was briefly air dried and re-suspended in SDS sample buffer containing 5% mercaptoethanol. 5 µl sample was applied to each well of a NuPAGE 4-12% acrylamide Bis-Tris gel (Invitrogen, San Diego, CA). The proteins were separated at a constant 200 volts for 35 minutes using MES running buffer. The gel was stained with 0.1% coomassie brilliant blue solution for 3 hours and then de-stained overnight. For western blotting of patient urine samples, 100 µl thawed urine was mixed with 1 ml acetone-methanol (1:1) and placed on ice for 1 hour for precipitation, gathered by centrifugation and re-suspended in SDS sample buffer containing 5% 15 ACS Paragon Plus Environment


mercaptoethanol.

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After SDS-PAGE, the urine proteins and pre-stained molecular weight

markers were transferred to nitrocellulose membranes for 6 minutes using iBLOT (Invitrogen). The nitrocellulose membrane was briefly washed with water and the non-specific sites blocked with LI-COR block solution (LI-COR Biosciences, Lincoln, NE). Urinary albumin and NGAL were visualized by incubation with 1/2000 dilution of rabbit anti-human serum albumin (Abcam, Cambridge, UK) and 1/500 dilution of goat anti-human NGAL (R&D Systems, Minneapolis, MN) in LI-COR block buffer containing 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO) overnight. The membrane was then washed three times with phosphate-buffered saline containing 0.05% Tween-20 followed by incubation with 1/10,000 dilutions each of Donkey anti-rabbit IgG 680 and Donkey anti-goat IgG 800 (LI-COR Biosciences). After 1 hour, the membrane was washed four times with phosphate-buffered saline containing 0.05% Tween-20 and visualized using an Odyssey-Fc (LI-COR Biosciences). Protein microarray protocols.

Commercial protein microarray chip was purchased from

RayBiotech (Custom G-Series Antibody Array, AAX-CUST-G).

Antibodies were printed on a

glass slide with 4 subarrays available per slide. The slide was blocked by 1× blocking buffer (0103004-B) for 30 minutes. The eluted urine samples were added into each sub-well of the microarray chip for 2 hours incubation at room temperature. The chip was then washed thoroughly with 1× wash buffers (0103004).

70 µl of 1× biotin-conjugated anti-cytokines were

added to each subarray and the chip was incubated in room temperature with gentle shaking. After 2 hours, the chip was washed and 70 µl of streptavidin-CW800 (100 ng/ml in 1X blocking buffer) was added and incubated in dark for 20 mins. The chip was washed thoroughly with wash buffer then nanopure water and blow dried under nitrogen gas. The glass chip was scanned by Licor Odyssey CLx scanner using 800 nm channel (intensity=2, resolution=21µm, scanning height=1mm). Median background signal was adopted for analysis spot intensity. CD spectroscopy. The CD measurements were performed using a spectropolarimeter JASCO J-810. The spectrum was collected at the rate of 20 nm per minute at a response time of 16 16 ACS Paragon Plus Environment

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seconds. Before CD measurement, the ZIF-8 encapsulated HSA was first eluted and then filtered to remove any ZIF-8 byproduct using centrifuge tube with 30 kDa filter.

The HSA

recovered from various treatments was quantified by BCA assay for calculating molar ellipticity. The secondary structures of HSA (α-helical content, β-sheet content) were analyzed using CDPro software from CD spectra. Paperfuge. The paperfuge was composed of a paper disc, a string and glass capillaries (microhematocrit capillary tubes, D=1.55 mm, Fisher Scientific). Common wood was used for the handles. The string was immobilized through paper disc using epoxy. After drawing blood, one end of capillary was sealed by capillary tube sealing tray (Thomas Scientific).

Supporting Information SEM images of bare paper and pure ZIF-8; TEM images of calcinated ZIF-8; Study of encapsulation, elution and preservation efficiency; Protein quantification with BCA assay and ELISA; CD experiment for studying protein structure; Western blot of patient samples; SDS-PAGE of eluates from dried serum samples; Study of patient urine pH after mixing with ZIF-8 precursors. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements

We acknowledge support from Air Force Office of Scientific Research (FA9550-15-1-0228) and National Institutes of Health (R01 CA141521). The authors thank the Nano Research Facility (NRF) at Washington University for providing access to electron microscopy facilities.



Figures

Figure 1. Schematic illustration depicting the concept of using MOF encapsulation for biospecimen preservation. By combining MOF encapsulation and dry spot sample collection, protein biomarkers can be preserved on a paper substrate under non-refrigerated conditions. Before bioanalysis, the MOF-encapsulated proteins can be recovered without losing structure and function.


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Figure 2. (A) Schematic illustrating the sample preparation of ZIF-8 encapsulated biofluid on a paper substrate. SEM image of a paper substrate after drying NGAL-spiked artificial urine (B) with and (C) without ZIF-8 precursors. (D) SEM image of NGAL-embedded ZIF-8 crystals showing uniform size and shape. Inset: TEM image of a typical NGAL-embedded ZIF-8 crystal. Scale bar: 200 nm. (E) XRD pattern of pure ZIF-8 and NGAL-embedded ZIF-8 crystals. (F) FTIR spectra of NGAL, pure ZIF-8 and NGAL-embedded ZIF-8 crystals. (G) Raman spectra of NGAL, pure ZIF-8 and NGAL-embedded ZIF-8 crystals.



Figure 3. (A) Preservation efficacy of NGAL with and without ZIF-8 encapsulation. NGAL added on a paper card was stored at 25, 40 or 60°C for one week. The ZIF-8 encapsulation shows comparable preservation, after storage at either room temperature or elevated temperatures, to the refrigeration method (freezing liquid samples at -20°C). (B) Preservation efficacy of NGAL on paper card at 25, 40 or 60°C for different durations. Results are the mean and standard deviation from three independent samples. (C) Preservation efficacy of NGAL in urine from three acute kidney injury patients with and without ZIF-8 encapsulation on paper cards stored at 25 or 40°C for one week. Results are the mean and standard deviation from three independent samples. (D) Fluorescence intensity maps (generated from protein microarray) of #3 patient’s urinary biomarkers with and without ZIF-8 encapsulation on paper cards stored at 25 or 40°C for one week. Frozen liquid sample (-20°C) was used as a reference. (E) Plot depicting the fluorescence intensity of various biomarkers for the samples described in (D). POSs represent 3 distinct Positive Control signal intensities (POS1>POS2>POS3). Results are the mean and standard deviation from two parallel dots for each biomarker.


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Figure 4. (A) Preservation efficacy of CA-125 spiked in serum with different dilutions. Storage condition: 40°C for one week. (B) XRD patterns of ZIF-8 crystals formed in the serum with different dilutions. (C) SEM images of ZIF-8 crystals formed in 5- and 20-fold diluted serum. (D)-(F) Preservation efficacy of CA-125 spiked in serum, heparin-plasma or EDTA-plasma on paper card at 25, 40 or 60°C for different durations. Results are the mean and standard deviation from three independent samples. Within four weeks, the ZIF-8 encapsulation shows comparable preservation, after storage at 25 and 40°C, to the refrigeration method (freezing liquid samples at -20°C).



Figure 5. (A) The procedure of preserving protein biomarkers in fresh blood samples by combining hand-powered centrifuge (“paperfuge”) and MOF-based preservation. (B) Preservation efficacy of CA-125 spiked in blood under temperature fluctuations in lab. Storage condition: two days 25°C followed by two days 40°C as one cycle and run for two cycles. (C) Preservation efficacy of CA-125 spiked in blood after 10 days of unknown shipping and handling conditions. The samples were mailed from Missouri to California and sent back to Missouri using regular mailing service. Results are the mean and standard deviation from three independent samples.


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