App-based quantification of crystal phases and amorphous content in ZIF biocomposites

The performance of zeolitic imidazolate frameworks (ZIFs) as protective hosts for proteins in drug delivery or biocatalysis strongly depends on the type of crystalline phase used for the encapsulation of the biomacromolecule (biomacromolecule@ZIF). Therefore, quantifying the different crystal phases and the amount of amorphous content of ZIFs is becoming increasingly important for a better understanding of the structure–property relationship. Typically, crystalline ZIF phases are qualitatively identified from diffraction patterns. However, accurate phase examinations are time-consuming and require specialized expertise. Here, we propose a calibration procedure (internal standard ZrO2) for the rapid and quantitative analysis of crystalline and amorphous ZIF phases from diffraction patterns. We integrated the procedure into a user-friendly web application, named ZIF Phase Analysis, which facilitates ZIF-based data analysis. As a result, it is now possible to quantify i) the relative amount of various common crystal phases (sodalite, diamondoid, ZIF-CO3-1, ZIF-EC-1, U12 and ZIF-L) in biomacromolecule@ZIF biocomposites based on Zn2+ and 2-methylimidazole (HmIM) and ii) the crystalline-to-amorphous ratio. This new analysis tool will advance the research on ZIF biocomposites for drug delivery and biocatalysis.

ZIF-L 1 : 20 ml of an aqueous 0.05 mM Zn(NO 3 ) 2 *6(H 2 O) Solution was added to 20 ml of an aqueous 0.4 mM 2mIm Solution. The reaction was stirred for 4 h. The solid product was separated via centrifugation (13000 rpm for 5 min; centrifuge used: Eppendorf 5425) and the supernatant was discarded. The powder was washed 3 times with 20 ml each DI water and then air-dried for 48 h at room temperature (23° C). For the calibration of each phase, mixtures with varying content of the pure phase with a constant amount of an internal standard (ZrO 2 ) were prepared. 5 mg of ZrO 2 were suspended in 150 µl DI water, sonicated for 15 minutes and added to a varying amount of pure ZIF phase (0,1 mg; 0.5 mg; 1 mg; 3 mg; 5 mg). The suspensions were vortexed for 1 minute to ensure a homogeneously mixed suspension. From each suspension mixture 50 µl were drop-cast on a 1.5 cm x 1.5 cm piece of Si and air-dried for 48 h at room temperature (23° C). Triplicates were made from each mixture. S3.2: Quantification Peaks and Reference Intensity Ratios (RIRs):  4 selected ZIF biocomposite samples (S1-S4) with different phases (U12, Dia, ZIF-C and Sod, respectively) were made according to literature. For this, stock solutions of Zn(OAc) 2 *2(H 2 O) (160 mM), 2mIm (2560 mM) and BSA (70 mg/ml) were diluted and mixed in varying ratios to yield a final reaction volume of 2 ml. The precise recipe can be found in Table S2. The reaction mixtures were left at static conditions at room temperature (23°C) for 24 h. The solid product was separated via centrifugation (13000 rpm for 5 min; centrifuge used: Eppendorf 5425) and the supernatant was discarded. The powder was washed either 3 times with 1 ml each DI water (water washing) or 2 times with 1 ml DI water and subsequently 2 times with 1 ml ethanol (ethanol washing). Finally, the powder was air-dried for 48 h at room temperature (23° C).  Figure S4: FT-IR spectra of the synthesised ZIF biocomposite Samples (S1-S4) with BSA in the 4 different phases (U12, Diamondoid, ZIF-C and Sodalite) The bands corresponding to the amid bonds located at 1700-1610 cm -1 and 1595-1480 cm -1 in BSA are very prominent in the composites. 8,9 Moreover, the peaks at 420 cm -1 and 427 cm -1 , assigned to the Zn-N stretching in sod (as well as dia, U12, ZIF-EC-1 and ZIF-L) and ZIF-C respectively, are distinct in the spectra. 2,4,10 Depending on the reaction conditions and washing, ZIF-C can be obtained. This can be seen if additional modes are present in the region of 700 to 850 cm -1 and 1300 to 1400 cm -1 , which are due to stretching modes of the incorporated CO 3 2-. 4 Through FT-IR spectroscopy one can therefore get a first impression: if the protein is encapsulated and if ZIF-C is formed.

S5: Phase and Amorphous Content Quantification
If the reference intensity ratio (RIR) of a material or phase is known, the weight fraction (wt%) of this material or phase (W i ) in a mixture can be determined. This is achieved by comparing the intensity of the most intense peak of the diffraction pattern of the sample (I i ) to the most intense peak of the diffraction pattern of a standard material (I c ), which is usually Al 2 O 3 (Corundum). The weight fraction of the standard material (W c ) should be known. This is summarised in the following equation. 11 = * * If Al 2 O 3 (Corundum) is not present in the mixture or not available, another reference material (like ZrO 2 ) can be added as an internal standard (IS). However, the change in the RIR has to be accounted for. A new adapted RIR (RIR i,IS ) arises, which is a combination of the RIR of the sample and the RIR of the internal standard. One can easily determine the RIR i,IS by mixing a known amount of the IS with a known amount of the sample. The following formula is then applied. The expected crystallinity should be 100 %. However, if this is not met, it means that a certain amount of the material does not contribute to the intensity and is therefore amorphous. 12

S6: Phases Selection
For each crystal phase we have identified 3 to 4 reference peaks (Table S3) that are used to uniquely identify the presence of a phase in a diffractogram. Each phase has a quantification peak, which is highlighted in bold in Table S3. It is the most intense peak and typically the first to appear when the phase concentration is low in the sample. The remaining peaks tend to appear as the phase concentration increases in the sample. Additionally, the quantification peak should not overlap with peaks from other phases.  The ZIF Phase Analysis app identifies all the peaks in a diffractogram using the diffractometry package 13 and then retain only those peaks that are in the vicinity of the reference peaks. In particular, each identified peak is compared to each reference peak by looking at the difference between the position of the identified peak and the angle of the reference peak. If the absolute difference is 0.10 or smaller, the identified peak is associated to the reference peak. The result is a table similar to Table S3 containing information about the angles of the retained peaks. Based on the retained peaks, the crystal phases present in a sample are determined using the following selection criteria:

Reference peaks (2θ, (hkl))
1. if only one of the quantification peaks (e.g. Dia peak 15.57°) is identified, then it is assumed that only one crystal phase is present in the sample (e.g. Dia) and the quantification peak is used to quantify the crystal phase; 2. if more than one quantification peak is identified, it is assumed that multiple crystal phases exist in the sample. To select the multiple phases present in a sample we developed an optimized procedure that minimizes the discrepancy between app-based and expert-based phase selection. The optimized procedure selects the Sod, Dia, and/or ZIF-C phase if the corresponding quantification peaks are identified. For the remaining phases, the presence of both the quantification peak and the majority (i.e. 3 out of 4, or 2 out of 3) of the reference peaks is required. Finally, the selected phases are quantified using the corresponding quantification peaks.
It is noted that there exists an overlap between two reference peaks of the Sod phase (7.36° and 18.07°) and the ZIF-L phase (7.33° and 18.00°). To account for this overlap, the ZIF-L phase is considered to be present in the sample only if the peak at 7.77 is identified as well.

S7: Limit of detection
The lowest ZIF wt% experimentally investigated to build the calibration curves was 2% (Fig. S3). Using a 3% wt/wt ZIF-L/ZrO 2 , the ZIF-L (the sample with the lowest RIR and therefore the lowest diffraction intensity compared to ZrO 2 ) diffraction peaks were barely detectable (Fig. S7c,d). Conversely, using the same acquisition parameters (i.e. Cu 9kW source, 4°/min), the quantification peak(s) of the other ZIF phases were clearly identified. We investigated additional wt% (i.e. 1, 0.5, 0.1) of the ZIF phase with the highest RIR (sodalite) to determine its limit of detection in a mixture with ZrO 2 . We found that 0.5% wt/wt sod/ZrO 2 is the lowest sod wt% detectable using the same acquisition parameters of the other samples (Fig. S7a,b). However, to ensure the reproducibility of the results for all the phases, we suggest not to use a ZIF wt% lower than 2% when preparing the samples for the calibration curve.