Hofmeister Effect Promoted the Introduction of Tunable Large Mesopores in MOFs at Low Temperature for Femtomolar ALP Detection

Abstract In addressing the demand for hierarchically mesoporous metal‐organic frameworks (HMMOFs) with adjustable large mesopores, a method based on the synergistic effects of low‐temperature microemulsions and Hofmeister ions is developed. Low temperature dramatically enhanced the solubility of hydrophobic solvent in the microemulsion core, enlarging the mesopores in HMMOFs replica. Meanwhile, Hofmeister salt‐in ions continuously controlled mesopore expansion by modulating the permeability of swelling agent into the microemulsion core. The large mesopores up to 33 nm provided sufficient space for the alkaline phosphatase (ALP) enrichment, and retained the remaining channel to facilitate the free mass diffusion. Leveraging these advantages, a colorimetric sensor is successfully developed using large‐mesopore HMMOFs for femtomolar ALP detection based on the enrichment and cycling amplification principles. The sensor exhibited a linear detection range of 100 to 7500 fm and a limit of detection of 42 fm, presenting over 4000 times higher sensitivity than classic para‐nitrophenyl phosphate colorimetric methods. Such high sensitivity highlights the importance of adjustable mesoporous structures of HMMOFs in advanced sensing applications, and prefigures their potential for detecting large biomolecules in diagnostics and biomedical research.

Studies were recorded with an RF-6000 spectro-fluorophotometer (Shimadzu).The dynamic light scattering (DLS) was measured with a ZEN3700 system.For the detection of ALP based on the colorimetric array, Thermo scientific microplate reader was used to determine the absorption value of each sample. .It could be observed that no mesopores were formed in the MOFs when salting-out ions such as SO 4  2-and HPO 4 2-were employed (Figures S1B and C).When no salt-in ions were added, almost no mesopores appeared on the surface of HMUiO-66(Ce) (Figure S1D).

S3. Results and Discussion
Upon introducing salt-in ions, as the salt-in capability increased (in the order NO 3 -< I -< ClO 4 -), the quantity and orderliness of the mesopores in the HMUiO-66(Ce) gradually improved (Figures S1E-G).This can be attributed to that the salt-in ions enhanced the interaction between the MOFs and micelles, thereby promoting the growth of MOF crystals on the micelle surface and leading to the formation of an ordered mesoporous structure.On the other hand, while NaSCN possess higher salt-in capabilities, it would decompose under acidic conditions to produce the highly toxic HCN gas.Therefore, NaClO 4 was the preferred choice as the salt-in ion for the synthesis of mesoporous MOFs.Table S3.Textural parameters for the HMUiO-66(Ce) samples synthesized at different temperatures using toluene/F127 microemulsions.In the experiment, the molar feed ratios of toluene/F127 and NaClO 4 •H 2 O/F127 were kept constant at 455 and 420, respectively.HMUiO-66(Ce).Sample standard deviation (σ) for the blank sample, without the addition of ALP was calculated to be 0.00333.The slope value was taken from a calibration curve for absorbance intensity against ALP concentration, which was calculated to be 2.4×10 -4 (Figure S10).fM, 5×10 6 fM, 1×10 7 fM, 1.5×10 7 fM, 2×10 7 fM and 5×10 7 fM, respectively.Sample standard deviation (σ) for the blank sample, without the addition of ALP was calculated to be 0.00591.The slope value was taken from a calibration curve for absorbance intensity against ALP concentration, which was calculated to be 6.9×10 -6 (Figure S14).Sample standard deviation (σ) for the blank sample, without the addition of ALP was calculated to be 0.00403.The slope value was taken from a calibration curve for absorbance intensity against ALP concentration, which was calculated to be 6.5×10 -8 (Figure S15).  between different literatures.The activity unit (U/L) = (C ALP × M ALP )* 38000 C ALP is the concentration unit of ALP (M); M ALP is the relative molecular weight of ALP, which is around 86000. [24]tection of ALP Activity Concentration in Human Serum Samples using the

Commercialized ALP Detection Kit
The human serum samples, provided by Renji Hospital, Shanghai Jiaotong University School of Medicine, were collected from four volunteers.We detected the ALP activity concentration in human serum samples according to the instructions of the commercial ALP detection kit.The kit contains detection buffer, colorimetric substrate of PNPP solution, pnitrophenol standard solution (10 mM), and reaction termination solution.The ALP activity concentration is defined as 1 U/L, which is the amount of alkaline phosphatase needed to hydrolyze 1 μM of the PNPP colorimetric substrate to produce 1 μM p-nitrophenol per min in a diethanolamine buffer of pH 9.8 at 37 o C.
First, we conducted a standard curve test for p-nitrophenol, which is the hydrolysis product of PNPP.Specifically, 10 μL of the p-nitrophenol standard solution (10 mM) was diluted with the detection buffer to obtain a p-nitrophenol solution with a concentration of 0.5 mM.Subsequently, it was further diluted to different concentrations of p-nitrophenol solution (0 μM, 20 μM, 40 μM, 80 μM, 120 μM, 160 μM, 200 μM).Then, 100 μL of the above pnitrophenol solutions was added to a 96-well plate, followed by the addition of 100 μL of reaction termination solution.The absorbance at 405 nm was measured using a microplate reader.The enhanced absorbance values were obtained by subtracting the absorbance of the blank sample from the absorbance of p-nitrophenol samples at different concentrations.These enhanced absorbance were linearly fitted against the different p-nitrophenol concentrations to establish the standard curve for p-nitrophenol, the hydrolysis product of PNPP.Finally, a linear fit was performed on these enhanced absorbance values versus the p-nitrophenol concentrations, generating the standard curve for the p-nitrophenol (Figure S16).
Then, ALP detection in human serum was carried out utilizing the commercial ALP detection kit.Each serum sample was assayed in four parallel setups.In the 96-well plate, 50 μL of detection buffer and 50 μL of colorimetric substrate solution were mixed as the blank control groups.Additionally, 10 μL of serum sample was added to a mixture of 40 μL detection buffer and 50 μL colorimetric substrate solution, resulting in 10-fold diluted serum samples.After the colorimetric reaction at 37 °C for 10 min, 100 μL of reaction termination solution was added to both the blank control groups and the serum sample groups.
Absorbance at 405 nm was measured using a microplate reader after the colorimetric reaction.
The enhanced absorbance values were obtained by subtracting the absorbance of the blank sample groups from the absorbance of serum sample groups.According to the p-nitrophenol standard curve, the concentrations of produced p-nitrophenol were calculated using the following formula (1): (1) Where represents the enhanced absorbance values at 405 nm for serum sample groups after the colorimetric reaction.
Finally, the activity concentrations of serum samples were calculated using the following formula (2): ( Where

Figure S1 .
Figure S1.(A) Schematic diagram of traditional Hofmeister series.(B-G) SEM images of

Figure S2 .
Figure S2.(A-E) SEM and (F-J) their corresponding enlarged images in the red selection area

Figure S5 .
Figure S5.(A) N 2 sorption isotherms and (B) their corresponding pore size distribution

Figure S6 .
Figure S6.(A) N 2 sorption isotherms and (B) their corresponding pore size distribution

Figure S7 .
Figure S7.XRD patterns for HMUiO-66(Ce) samples synthesized at 40 o C and 11 o C after

Figure S10 .
Figure S10.Relation of absorbance intensity against ALP concentration based on HMUiO-

Figure S12 .
Figure S12.The absorbance change at 405 nm plotted against ALP concentrations using the

Figure S13 .
Figure S13.The absorbance change at 570 nm plotted against ALP concentrations using

Figure S14 .
Figure S14.Relation of absorbance intensity against ALP concentration using single cyclic

Figure S15 .
Figure S15.Relation of absorbance intensity against ALP concentration using the classic C p-nitrophenol (μM) represents the concentration of p-nitrophenol produced by each serum sample after the colorimetric reaction within 10 min, t (10 min) represents the colorimetric reaction time, and N (10-fold) represents the dilution fold of the serum sample.Detection of ALP Activity Concentration in Human Serum Samples using the DevelopedMethodFirst, we prepared standard solutions with different ALP concentrations (0 fM, 100 fM, 250 fM, 500 fM, 750 fM, 1000 fM, 2500 fM, 5000 fM), corresponding to ALP activity concentrations of 0 U/L, 0.0003268 U/L, 0.000817 U/L, 0.001634 U/L, 0.002451 U/L, 0.003268 U/L, 0.00817 U/L, and 0.01634 U/L.The ALP activity concentration standard curve was tested using these different ALP standard solutions (FigureS17), based on the standard colorimetric detection procedure of ALP in the presence of HMUiO-66(Ce).The notable variation was the use of a higher concentration of HMUiO-66(Ce) to meet the requirements for enriching ALP in serum samples, where 50 μL of HMUiO-66(Ce) suspension (250 mg/L, in ethanol) was added to the bottom of each tube.Then, ALP in human serum was detected by using the developed method.Four parallel samples were set up for each human serum sample.The human serum sample was diluted 10 5 -fold with Tris-HCl buffer (20 mM, pH 7.4), and then the standard colorimetric detection procedure of ALP in the presence of HMUiO-66(Ce) was conducted.Absorbance at 570 nm was measured using a microplate reader after the colorimetric reaction.The enhanced absorbance of the serum sample group was obtained by subtracting the absorbance of the blank sample groups from the absorbance of serum sample groups.Based on the standard curve of ALP activity concentration, the ALP activity concentration in human serum was calculated using the following formula (3): (3) Where represents the enhanced absorbance values at 570 nm for serum sample groups after the colorimetric reaction, and N (10 5 -fold) represents the dilution fold of the serum sample.The spiked recovery tests were conducted by additional adding three different ALP concentrations (100 U/L, 300 U/L and 600 U/L) into the serum sample 1, in which already contains 105.3 U/L of ALP as determined by the commercialized ALP detection kit.Then, the total ALP concentration was measured from the prepared spiked samples in four parallel setups based on the developed method.The recovery was calculated (Recovery = detected concentration of total ALP/actual concentration of total ALP × 100).

Figure S16 .
Figure S16.The fitting standard curve for the enhanced absorbance values against p-

Figure S17 .
Figure S17.The fitting standard curve for the enhanced absorbance values against (A) ALP

Table S1 .
Textural parameters for the representative HMUiO-66(Ce) samples synthesized at different temperatures.

Table S4 .
Comparison of the performance for ALP activity detection based on the recent developed colorimetric and fluorometric methods.

Table S5 .
The comparison of detected ALP in human serum samples from four volunteers by using the commercialized ALP detection kit and our developed method.

Table S6 .
Recovery of the additional added ALP in serum samples determined by the developed method.The serum sample 1 was used to added with extra ALP, in which already contains 105.3 U/L of ALP as determined by the commercialized ALP detection kit. a