Preparation of functionalized mesoporous silica nanoparticles as mass tags for application in mass cytometry

Abstract

Functionalized niesoporous silica nanoparticles (MSNs) and methods for making functionalized MSNs are provided. MSNs are synthesized and post-modified with zwitterionic silanes and polyethylene glycol (PEG) silanes to suppress non-specific protein adsorption. The PEG silane may be a long-chain PEGsk-silane and the zwitterionic silane may be a zwitterionic sulfobetaine silane. The MSNs may be loaded with at least one lanthanide ion to serve as a mass tag reagent for mass cytometry. The MSNs may be conjugated with an antibody; for example, the MSN may comprise an azide-PEGsk-silane conjugated with a dibenzocyclooctyne-functionalized anti-biotin antibody (DBCO-anti-biotin Abs).

Description

[0001]Attorney Docket No.: 4637-0019WO01 Preparation of Functionalized Mesoporous Silica Nanoparticles as Mass Tags for Application in Mass Cytometry 5 BACKGROUND In the field of modern biomedical research, understanding the complexity of cellular biology, disease mechanisms, and immune responses has never been more critical. To address these issues, scientists have been developing innovative technologies to identify cellular biomarkers that can provide deeper insights into the diverse and complex world of biology. 10 Among these breakthrough technologies, mass cytometry (MC) has emerged as a powerful tool with the potential to revolutionize single cell analysis. MC is a cutting-edge bioanalytical technology that integrates the principles of flow cytometry with atomic mass spectrometry, enabling researchers to measure multiple parameters simultaneously at the single-cell level. In this approach, cells are first stained with heavy-metal 15 isotope-labeled antibodies (Abs). Subsequently, the single-cell suspension is introduced into an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOF-MS), enabling the examination of multiple target biomarkers by monitoring the signals in different and discrete mass channels. By replacing traditional fluorophores with heavy-metal isotopes as Ab tags, MC overcomes several limitations inherent to flow cytometry, such as the interference from spectral 20 overlap and limited multiplexing capacity. In principle, MC has the capability to concurrently detect more than 100 parameters within an individual experiment, determined by the number of various stable heavy metal isotopes with masses in the range of m / z 75 to 209. However, due to the lack of carriers capable of capturing heavy metal isotopes, only about 50 parameters can currently be identified on a routine basis. Therefore, there is a long felt and growing demand for 25 the development of novel elemental mass tag reagents to expand detection channels and enhance characterization and multiplexing capabilities. BRIEF SUMMARY The MSN-based nanoparticles as disclosed herein provide in various embodiments high- 30 sensitivity mass tag reagents. The advantages of the disclosed MSN-based nanoparticles include, but are not limited to, low levels of nonspecific binding (NSB) and successful attachment with Attorney Docket No.: 4637-0019WO01 Abs with MSNs. In various embodiments, the conjugation of antibodies with PMSNs-Zwi- PEG5k-N3are advantageous for use as a biomarker for detection by mass cytometry. In various aspects and embodiments, provided are compositions comprising functionalized mesoporous silica nanoparticles, wherein the functionalized MSN comprises: mesoporous silica nanoparticles (MSN); at least one zwitterionic silane; and at least one long-chain polyethylene glycol (PEG) functionalized silane. In various embodiments, the at least one zwitterionic silane is a zwitterionic betaine group containing silane. In various embodiments, the at least one zwitterionic betaine group containing silane is a zwitterionic carboxybetaine silane. In various embodiments, the at least one zwitterionic betaine group containing silane is a zwitterionic sulfobetaine silane. In various embodiments, the long-chain polyethylene glycol (PEG) functionalized silane is a PEG5k-silane, and preferably a long-chain PEG silane. In various embodiments, the PEG5k-silane is an azide-PEG5k-silane. In various embodiments, the zwitterionic silanes has the following features: (a) one or more quaternary ammonium groups (-N⁺(CH₃)₃) or other cationic moieties, (b) one or more sulfonate (-SO₃⁻), carboxylate (-COO⁻), or phosphate groups, (c) a medium chain length (e.g., C3-C6), for example, to balance hydrophilicity and structural integrity, and (d) one or more hydrolyzable groups, such as, for example, trimethoxy- or triethoxysilane. As used herein, the term “long-chain” in the context of PEG refers to a PEG having a molecular weight of greater than about 1.5 kiloDaltons (kDa), greater than about 3 kDa, greater than about4 kDa, and in various embodiments a molecular weight in the range between about 3 kDa to about 10 kDa. In various aspects and embodiments, the functionalized MSN further comprise lanthanide ions, such as, for example, those selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof. In various embodiments, the lanthanide ion comprises at least about 1 x 104to at least about 7.4 x 104lanthanide ions per functionalized MSN. In various aspects and embodiments, provided are the functionalized MSN disclosed herein conjugated an antibody (Ab). In various embodiments, provided are compositions wherein the functionalized MSN is conjugated with dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs), and in various embodiments the functionalized MSN comprises an Attorney Docket No.: 4637-0019WO01 azide-PEG5k-silane conjugated with the dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs). In various aspects and embodiments the multi-functionalized mesoporous silica nanoparticles (MSN) disclosed herein are prepared through post-modification with zwitterionic silanes and, in various embodiments, long-chain PEG5k silanes. In various embodiments, the fictionalized MSN are prepared through post-modification with zwitterionic sulfobetaine silanes. The series of surface modifications of PMSNs disclosed herein, to enhance their colloidal stabilities in buffer solutions and reduce non-specific binding (NSB) with cells, provide for compositions suitable for mass cytometry applications. The as-synthesized PMSNs-Zwi-mPEG5kNPs, with a diameter of 48 nm, was and can be lyophilized and redispersed into both H2O and 1× PBS buffer solution with good stability. In various embodiments, these functionalized MSNs exhibited a remarkable capacity to carry high amounts of lanthanide ions (e.g., Tb, 19.6 mg / g), with negligible ion loss in H2O (~ 1.4 %) and 1× PBS buffer (~ 0.2 %). Furthermore, pretreatment of Tb@PMSNs-Zwi-mPEG5k NPs with PBS buffer for 20 min greatly reduced the leakage of Tb ions into various other buffer solutions (e.g., HEPES buffer, MES buffer, Bis-tris buffer) to less than 1 %, presumably by precipitation as TbPO4. These nanoparticles were discovered to be versatile for loading various lanthanide elements, thereby in various embodiments providing new elemental mass tags. In various aspects the functionalized MSN employ PEG5kchains. In various embodiments a PEG5k chain density of 0.52 chains per nm2on PMSNs-Zwi-mPEG5k showed the least extent of HSA adsorption (1.1 wt%), suggesting, without being held to theory, that this dense brush conformation was effective at suppressing protein adsorption. In various aspects, replacement of mPEG5k-silane with azide-PEG5k-silane was carried out to prepare PMSNs-Zwi-PEG5k-N3 NPs. These were then reacted with DBCO-modified anti-biotin antibodies to generate distinct NP-Ab conjugates. All these conjugates retained their Ab functionalities and were capable of binding target biotin Cy5 molecules. In various embodiments, the bioconjugation reaction was performed at 25°C for 4 h where a 50:1 Ab-to-NP ratio was observed to provide a high conjugation efficiency while preserving a large extent of Ab functionality. Attorney Docket No.: 4637-0019WO01 In various embodiments, the resulting methoxy-terminated nanoparticles (PMSN-Zwi- mPEG5k) exhibited the capacity to carry up to 7.4 × 104Tb ions per nanoparticle (NP), with negligible ion loss observed in both H2O and 1× PBS buffer. In various embodiments, leakage detected from Tb-loaded PMSN-Zwi-mPEG5k in various buffers is suppressed by first immersing the NPs in 1× PBS buffer. In various embodiments, provided are compositions and chain densities of mPEG5kon the surface of NPs, that provide for high grafting density and low non- specific binding to serum proteins. In various embodiments, provided are NPs (PMSN-Zwi- PEG5k-N3) with attached azide-PEG5k-silanes able to conjugate with dibenzocyclooctyne- functionalized anti-biotin Abs (DBCO-anti-biotin Abs) via click chemistry. In various embodiments, the purified NP-Ab conjugates were effective at binding biotin Cy5 molecules, as evidenced, for example, by the distinct peaks corresponding to Cy5 under UV-vis detection. In various aspects and embodiments, also provided are methods of analyzing a cell, the methods comprising introducing the cell to a composition comprising functionalized MSNs as disclosed herein. In various embodiments, a composition comprising functionalized MSNs for use in a method for analyzing a cell comprises a mixture of functionalized MSNs, the mixture comprising a plurality of functionalized MSNs, each functionalized MSN being an embodiment of a functionalized MSN as disclosed herein. In various embodiments, such mixtures comprise at least 10, 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 functionalized MSNs. In various embodiments, the functionalized MSNs of the mixture of a plurality of functionalized MSNs comprise: mesoporous silica nanoparticles (MSN); at least one zwitterionic silane; and at least one long-chain polyethylene glycol (PEG) functionalized silane. In various embodiments, the at least one zwitterionic silane is a zwitterionic sulfobetaine silane. In various embodiments, the long-chain polyethylene glycol (PEG) functionalized silane is a PEG5k-silane. In various embodiments, the PEG5k-silane is an azide-PEG5k-silane. In various embodiments, the functionalized MSN comprises zwitterionic sulfobetaine silanes and long-chain PEG5ksilanes. In various aspects and embodiments, at least a portion of the functionalized MSNs in the mixture of a plurality of functionalized MSNs further comprise lanthanide ions, such as, for example, those selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof. In various embodiments, the lanthanide ion is Tb Attorney Docket No.: 4637-0019WO01 and such functionalized MSNs have at least about 1 x 104to at least about 7.4 x 104Tb ions per functionalized MSN. In various aspects and embodiments, at least a portion of the functionalized MSNs in the mixture of a plurality of functionalized MSNs are conjugated with an antibody (Ab). In various embodiments, such functionalized MSNs are conjugated with dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs), and in various embodiments such functionalized MSNs comprises an azide-PEG5k-silane conjugated with the dibenzocyclooctyne-functionalized anti- biotin Abs (DBCO-anti-biotin Abs). BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein: FIG.1 is a schematic diagram of a procedure for preparing multifunctional MSNs. FIG.2 has multiple panels, where FIG.2A and FIG.2B are TEM images; FIG.2C and FIG.2D are size distribution histograms, and FIG.2E and FIG.2F are DLS results of redispersed PMSNs NPs (dh = 73.0 nm, PDI = 0.14) and redispersed PMSNs-Zwi-mPEG5kNPs (dh = 81.9 nm, PDI = 0.11). FIG.3 has multiple panels, where FIG.3A exemplifies the room temperature synthesis of zwitterion sulfobetaine silane (SBS), FIG.3B is a 1H NMR spectrum and FIG.3C is a 13C NMR spectrum of the as-synthesized SBS of Examples 1-13. FIG.4 has multiple panels, which are TEM images where FIG.4A shows PMSNs and FIG.4B shows PMSNs-Zwi-mPEG5kat higher magnification than FIGs.2A and 2B respectively. FIG.5 has multiple panels, wherein FIG. 5A is a digital picture of dried powder and redispersed colloidal solution (20 mg / mL) of PMSNs; FIG.5B shows the hydrodynamic diameter distributions of as-synthesized PMSNs solution (dh = 70.5 nm, PDI = 0.097) and redispersed PMSNs solution (dh = 73.0 nm, PDI = 0.14); FIG.5C shows the stability of redispersed PMSNs- Zwi-mPEG5k in H2O (hollow square symbols) and 1× PBS buffer (hollow circular symbols) at 4°C for 7 days; and FIG.5D shows the reproducibility test of the synthesis of PMSNs NPs. The error bars in FIG.5D represent one standard deviation in size distribution. FIG.6 has multiple panels, wherein FIG.6A shows the FTIR spectra of PMSNs NPs before and after CTAC removal, PMSNs-Zwi NPs, and PMSNs-Zwi-mPEG5k NPs; FIG. 6B Attorney Docket No.: 4637-0019WO01 shows the TGA curves of bare MSNs, PMSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG5k NPs; FIG. 6C shows nitrogen adsorption-desorption isotherms; and FIG. 6D shows the pore size distribution calculated by Barrett-Joyner-Halenda (BJH) method of PMSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG5k NPs. FIG. 7 shows the FTIR spectra of PMSNs of Examples 1-11 before CTAC template removal and after two extraction cycles in a mixed solution of concentrated HCl and ethanol. FIG. 8 shows the 1H NMR spectra (acquired on a 600 MHz Agilent DD2 NMR Spectrometer) of various NP suspensions; where in FIG.8 from bottom to top: PMSN NPs in D2O (20 mg / mL), PMSN-Zwi NPs in D2O (5 mg / mL), and PMSNs-Zwi-mPEG5kNPs in D2O (5 mg / mL). FIG.9 shows the loading content of various lanthanide elements on PMSNs-Zwi-mPEG5k NPs. A mixture of lanthanides at 1 mg / mL each was mixed with nanoparticles (0.5 mg / mL) for 24 h. The error bars represent one standard deviation and were calculated from three independent samples, with triplicate measurements on each sample. FIG.10 has multiple panels, where FIG, 10A shows the leaching profile of Tb3+ ions from PMSNs-Zwi-mPEG5kNPs in different aqueous environments without pretreatment with PBS buffer at various conditions: 0.1 M Na2CO3 / NaHCO3 buffer (pH 10.6), 0.1 M MES buffer (pH 5.5), 0.02 M Bis-tris buffer (pH 6.5), 0.01 M HEPES buffer (pH 7.4), H2O and 1× PBS; FIG. 10B shows a comparison of Tb3+ leaching profiles of PMSNs-Zwi-mPEG5kNPs in 0.1 M Na2CO3 / NaHCO3 buffer (pH 10.6) without pretreatment (solid line) and with pretreatment (dashed line); FIG.10C shows TEM images of PMSNs-Zwi-mPEG5k NPs before immersing in 0.1 M Na2CO3 / NaHCO3 buffer (pH 10.6) for 4 days; and FIG. 10D shows TEM images of PMSNs-Zwi-mPEG5kNPs after immersing in 0.1 M Na2CO3 / NaHCO3 buffer (pH 10.6) for 4 days. FIG. 11 has multiple panels, where FIG. 11A shows the leaching of Tb3+ ions from PMSNs-Zwi-mPEG5kNPs into 0.1 M MES buffer (pH 5.5) after different pretreatment times (i.e., 20 min, 40 min, 60 min and 120 min); FIG.11B shows the leaching profile of Tb3+ ions from PMSNs-Zwi-mPEG5k NPs in different aqueous environments after pretreatment in 1× PBS for 20 min at various conditions: 0.1 M Na2CO3 / NaHCO3 buffer (pH 10.6), 0.1 M MES buffer (pH 5.5), 0.02 M Bis-tris buffer (pH 6.5), 0.01 M HEPES buffer (pH 7.4). H2O and 1× PBS buffer profiles are without pretreatment. Attorney Docket No.: 4637-0019WO01 FIG. 12 has multiple panels, where FIG. 12A shows TGA curves of PMSNs-Zwi- mPEG5ksynthesized with different amounts of mPEG5k. The numbers behind the sample name in FIG.12A indicate the mass ratio of nanoparticles to mPEG5k, e.g., 1010 means the mass ratio of NP-to-mPEG5k is 10:10; FIG.12B shows the quantitative analysis of the grafted mPEG5k content, presented in terms of weight percentage (wt%) and grafting density (chains / nm2), derived from the TGA data displayed in FIG.12A; FIG.12C shows the quantitative analysis of adsorbed HSA of bare MSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG5k with different compositions. The error bars in FIG.12C represent one standard deviation calculated from three independent samples, with triplicate measurements on each sample; and FIG.12D shows the schematic illustration of PEG conformations at nanoparticle surface: brush-like (left) and dense brush-like conformation (right) for PMSNs-Zwi-mPEG5k. FIG.13 has multiple panels, where FIG.13A shows the calibration curve for a Pierce™ BCA protein assay used to quantify the amount of protein adsorbed onto nanoparticles; and FIG. 13B shows the calibration curve for a Micro BCATMprotein assay used to quantify the amount of anti-biotin Abs / control Abs labeled onto nanoparticles. FIG.14 shows a schematic diagram of a procedure for conjugating anti-biotin Abs to PMSNs-Zwi-PEG5k-N3 NPs and testing antigen recognition with Cy5 biotin molecules. FIG.15 has multiple panels, where FIG.15A is a TEM image of PMSNs-Zwi-PEG5k-N3 NPs prepared under substantially similar reaction conditions as for the preparation of PMSNs- Zwi-mPEG5k; FIG.15B shows the size distribution histogram of PMSNs-Zwi-PEG5k-N3 NPs (dTEM = 49.1 ± 3.1 nm, CV = 6.2%); FIG.15C shows the DLS results of PMSNs-Zwi-PEG5k- N3 NPs redispersed in PBS buffer (dh = 79.9 nm; PDI = 0.095) and PMSNs-Zwi-PEG5k-N3 NPs after storing in PBS buffer for 7 days (dh = 79.8 nm; PDI = 0.81); and FIG.15D shows the UV- vis spectra of PMSNs-Zwi-PEG5k-Cy5 NPs in 1× PBS buffer. FIG.16 has multiple panels, where FIG.16A is the UV−vis spectra of DBCO-modified anti-biotin Abs and DBCO-modified isotype control Abs (upper trace at A280). Each anti-biotin Ab and control Ab had approximately 4 DBCO groups; FIG. 16B shows the DLS results of samples in 1×PBS buffer: anti-biotin Abs ( dh = 10.7 nm, PDI = 0.036), PMSNs-Zwi-PEG5k-N3 NPs (dh = 79.9 nm, PDI = 0.095), NP-anti-biotin Ab conjugates (RT, 4h, 22:1, dh = 89.6 nm, PDI = 0.13) and NP-anti-biotin Ab conjugates (RT, 4h, 50:1, dh = 90.6 nm, PDI = 0.12); FIG.16C shows a UV−vis spectra of NP-anti-biotin Ab-Cy5, NP-control Ab-Cy5 and corresponding NP- Attorney Docket No.: 4637-0019WO01 anti-biotin Ab conjugates without adding Cy5 biotin (RT, 4h, 50:1). The dashed curve in FIG.16 C is a modeled baseline for NP-anti-biotin Ab-Cy5, obtained from a least squares regression of the corresponding NP-anti-biotin Ab spectrum that was then normalized to match the anti-biotin Ab-Cy5 trace. Inset: zoom in (500 to 800 nm); and FIG.16D shows the numbers of Cy5 per Ab after bioconjugation and purification under various conditions with Ab-to-NP feed ratios of 22:1 and 50:1. The error bars in FIG.16D represent one standard deviation determined from duplicate measurements on three independent samples. DETAILED DESCRIPTION Current elemental labeling strategies for the design of mass tag reagents are mainly based on metal-chelating polymers (MCPs) and various types of nanoparticles (NPs). In the case of MCPs, only a limited number of metals including Ln (La to Lu, except Pm), Y, In, Bi, Cd, Pt, Re and Te can be stably chelated onto the polymer sidechains and demonstrate good performance in MC. Despite significant efforts by researchers to incorporate a wider range of metal types into MCPs, substantial challenges persist, especially concerning polymer chain cross-linking and potential antibody-antigen recognition disruption. More importantly, the average metal atom count per polymer typically ranges from 20 to 25, resulting in 100-250 metal ions per Ab. This only allows for the effective detection of biomarkers expressed within the range of 104to 107biomarkers per cell, thereby limiting the lower threshold of MC measurements. In order to detect the low-abundance biomarkers (< 104copies per cell), like cytokine receptors, there is a need for ‘brighter’ mass tags that can carry more metal atoms. The present disclosure provides mesoporous silica nanoparticles (MSNs) as a basis for MC mass tags. MSNs are silica materials with pores that range in diameter from 2 to 50 nm. In various embodiments, the synthesis modifies 45 nm diameter MSNs and employs short-chain polyethylene glycol (PEG6-9 silane, PMSN) to promote colloidal stability is during preparation and the PMSNs were sequentially modified with zwitterionic sulfobetaine molecules and long- chain PEG5kpolymers (PMSN-Zwi-mPEG5k) to, e.g., reduce non-specific serum protein adsorption and facilitate Ab attachment. In various embodiments, the PMSN-Zwi-mPEG5k NPs were loaded with Tb ions in an aqueous solution, with negligible ion loss observed in 1× PBS buffer. In addition, also disclosed herein are embodiments with various compositions and chain densities of mPEG5k of PMSN-Zwi-mPEG5k NPs useful for example, in various embodiments Attorney Docket No.: 4637-0019WO01 and applications, to minimize non-specific binding of NPs to human serum albumin (HSA). In various embodiments, mPEG5ksilanes were replaced with N3-PEG5ksilanes to obtain N3- terminated NPs (PMSN-Zwi-PEG5k-N3). In various embodiments, the resulting PMSN-Zwi- PEG5k-N3 NPs were conjugated with DBCO-modified anti-biotin Abs via click chemistry, and in various embodiments, various bioconjugation conditions are provided; determined, for example, in various embodiments through recognition with biotinylated Cy5 molecules. Table 1 summarizes the physicochemical properties of various PMSN-Zwi-PEG5k NPs prepared and discussed herein. Table 1. Physiochemical Characterization of series of PMSN-Zwi-mPEG5k NPs. NP / PEG w / w dTEM(nm) Coefficient of Variation dh(nm) PDI (CV) 10:10 47.7 ± 2.8 5.8% 83.3 0.14 10:20 47.5 ± 2.6 5.6% 84.8 0.13 10:40 47.9 ± 3.1 6.5% 81.9 0.11 10:60 48.2 ± 2.4 5.0% 82.0 0.15 10:80 47.6 ± 2.6 5.5% 83.9 0.12 Preparation and Characterization of Multifunctional MSN NPs. To function as proficient MC reporters, MSN-based mass tag reagents need to fulfill several criteria. One criteria, for example, is the balance between the size of NP and the content of loaded lanthanides. If the nanoparticle size is too small it may not be capable of carrying sufficient lanthanides, whereas if it’s too large it could overwhelm the MC instrument with excess lanthanides. Moreover, surface modifications are necessary to endow NPs with colloidal stability in phosphate-containing buffers, reduce non-specific interactions with cells, and facilitate the introduction of proper functional groups for Ab conjugation. In various aspects and embodiments provided herein, MSNs were synthesized by a hydrothermal method with triethanolamine (TEA) as base catalyst, cetyltrimethylammonium chloride (CTAC) as structure-directing agent, and Attorney Docket No.: 4637-0019WO01 tetraethylorthosilicate (TEOS) as silica precursor. The reaction was run at 95 °C, and a short hydrophilic PEG6-9silane (M = 459-591) was introduced via co-condensation to improve the colloidal stability and dispersibility of the silica NPs to provide small (dTEM= 45 nm), uniform (CV < 8%) and redispersible PEG6-9-modified MSNs; see, for example, Table 1. These NPs were then sequentially treated with 3-(dimethyl(3- (trimethoxysilyl)propyl)ammonio)propane-1-sulfonate, a zwitterionic sulfobetaine-silane (SBS), and a longer chain polyethylene glycol-silane (PEG5k, M = 5000), to enhance colloidal stability and to reduce protein adsorption in subsequent applications of these materials. This set of reactions is summarized in FIG. 1, and details are provided in the Examples, where FIG. 1 illustrates that in various embodiments,the NPs are treated with PEG6-9 silane as PMSNs via co- condensation, CTAC was was removed those PMSNs were treated subsequently with SBS as PMSNs-Zwi, and then those PMSNs-Zwi particles to which the methoxy-PEG5ksilane have been attached as PMSNs-Zwi-mPEG5kare contacted with a lanthanide (Ln), in this illustration Tb, to provide Tb@PMSNs-Zwi- mPEG5k. Transmission electron microscopy (TEM) images of PMSNs and PMSNs-Zwi-mPEG5k, along with histograms of the size distribution are shown in FIGs.2A-2D. These PMSNs exhibit a uniform discrete spherical morphology with a mean diameter of 44.5 ± 3.4 nm (CV = 7.7%). A wormlike mesoporous structure can be seen at higher magnification in the TEM image displayed in FIG.4A. In this example, after surface modification with SBS and mPEG5k, the morphology of the nanoparticles remained spherical in shape and uniform in size, with an average diameter of 47.9 ± 3.1 nm (CV = 6.5%). Additionally, the outer edges and wormlike mesopore structures became blurred after the introduction of long-chain PEG polymers due to the limited contrast of the surface-bound polymers (see FIG.4B). In various embodiments, hydrophilic polymers were incorporated to provide for lyophilization of the samples and redispersal of the NPs. The redispersed PMSNs and PMSNs- Zwi-mPEG5kin H2O are optically transparent colloidal solutions, and the hydrodynamic size of the dried and redispersed PMSNs is almost the same as that of the as-synthesized PMSNs solution (see, e.g., FIG. 5B). FIGs. 2E and 2F present dynamic light scattering (DLS) results from redispersed samples of this example. They show a relatively narrow monomodal size distribution with a z-average diameter dh = 73.0 nm (PDI = 0.14) for PMSNs and 81.9 nm (PDI = 0.11) for Attorney Docket No.: 4637-0019WO01 PMSNs-Zwi-mPEG5k in H2O. The dh values of both samples in PBS buffers closely matched their dhvalues in H2O. As expected, dhvalues measured by DLS were larger than dTEMvalues because of the contribution of the hydration layer in the aqueous environment. The thickness of the hydration layer (Lh) associated with PEG5k corona can be estimated as ^^ ൫ௗ^^ =^ିௗಶಾ൯ ^ ଶ ≈ ଶ(82 − 48) = 17 ^^^^ (1)Neither the DLS their good stability in an and there was no obvious change in the hydrodynamic size during one-week storage of these nanoparticles in PBS buffer (FIG.5C). To assess the reproducibility of PMSNs synthesis, six batches of nanoparticles were prepared under the same conditions and the sizes were analyzed by TEM. As depicted in FIG.5D, all six nanoparticle samples (trial numbers) had a mean diameter of about 45 nm. Fourier transform infrared (FT-IR) spectroscopy was used to confirm removal of the CTAC template as well as to characterize subsequent surface modifications with SBS and mPEG5k. As shown in FIGs.6A and 7, before extraction of CTAC, two peaks attributed to C-H stretching vibrations can be seen at 2924 cm-1and 2855 cm-1. These peaks largely decreased after the first extraction cycle (12 h in an acidic ethanol solution), indicating successful removal of the CTAC template. However, even after the two cycles of extraction, there are still two peaks associated with -CH2 groups present (FIG.7). These can be attributed to the -CH2 groups in PEG6-9 silane incorporated into PMSNs during the co-condensation step. In various embodiments, the successful functionalization of SBS and mPEG5k onto the PMSNs surface was validated by the appearance of the quaternary ammonium group (-N+(CH3)2R-) at 1486 cm-1and the larger peak at 2924 cm-1and 2855 cm-1associated with the -CH2stretches of the PEG5kchains (see FIG.6A). In various embodiments, the silica framework of the multifunctional MSN-based nanoparticles remains substantially unchanged upon polymer coating, as shown by the preservation of peaks at 449 cm-1(Si-O bending), 796 cm-1(Si-O-Si symmetric stretching) and 1055 cm-1(Si-O-Si asymmetric stretching). The surface modification was also characterized by1H NMR as shown in FIG 8 and discussed in the Examples. Thermogravimetric analysis (TGA) was employed to quantify the extent of surface modification of PMSNs (see FIG.6B, note that the percentages shown by each trace in FIG.6B Attorney Docket No.: 4637-0019WO01 refer to percentage of mass retained). Bare MSNs without any modifications served as a baseline. Over a temperature range of 100 to 800 ^C, the bare NPs lost 7.4% of their mass (i.e. retained 92.6% of their mass). This loss of mass is believed to be mainly due to the removal of absorbed water and the dehydroxylation of silanol groups. The weight loss for PMSNs was found to be more significant at 23.2%, and attributed to the degradation of low-molecular-weight PEG6-9 silanes. The PMSNs-Zwi and PMSNs-Zwi-mPEG5k samples exhibited even greater weight losses at 28.5% and 41.1%, respectively, indicating the presence of approximately 5.3 wt% SBS and 12.6 wt% mPEG5k in the PMSNs-Zwi-mPEG5k NPs. Without being held to theory, this analysis shows that in various embodiments the zwitterionic sulfobetaine forms only volatile products upon pyrolysis and indicates the successful immobilization in the present disclosures of SBS and mPEG5k silane onto the surface of PMSNs nanoparticles. Nitrogen adsorption / desorption isotherms of PMSNs, PMSNs-Zwi, PMSNs-Zwi-mPEG5k samples in this example are displayed in FIG.6C and the results are presented in Table 2. All the adsorption / desorption plots are typical type IV isotherms, indicating a well-defined mesoporous structure. The sharp adsorption step at relative pressure (P / P0) in the range of 0.2-0.4, is a characteristic of mesoporous materials with relatively small pores. The a secondary adsorption step at higher P / P0> 0.8, is attributed here, without being held theory, to the interspace formed among the nanoparticles after freeze-drying called textural porosity. Additionally, it was determined that the capillary condensation behavior within mesopores of the samples of this example is reversible, resulting in a hysteresis loop in the isotherms. It was also determined that upon surface modification, the adsorption step becomes less steep compared to that of PMSNs, and the inflection point shifts to somewhat lower P / P0, suggesting a slight decrease in pore diameter as revealed by the pore size distribution curves in FIG.6D and Table 2. Thus in various embodiments, surface modification of PMSNs with SBS and mPEG5ksilane was used to adjust the surface area, pore volume, and pore diameter and provide a gradual reduction from 637, 514 to 229 m2 / g, 0.96, 0.66 to 0.43 cm3 / g, 2.7, 2.4 to 2.2 nm, respectively, as illustrated, e.g., in Table 2. Table 2. Summary of surface areas, pore volumes and pore sizes for various NP samples. Attorney Docket No.: 4637-0019WO01 Pore volumebSample Surface Areaa(m2 / g) Pore sizec(nm) (cm3 / g) PMSNs 637 0.96 2.7 PMSNs-Zwi 514 0.66 2.4 PMSNs-Zwi- mPEG5k 229 0.43 2.2aSpecific surface area was calculated from data in the range P / P0 < 0.3 using the BET method (Brunauer, S.; Emmett, P. H.; Teller, E., “Adsorption of Gases in Multimolecular Layers”, Jbournal of the American Chemical Society 1938, 60 (2), 309-319). Total pore volume was calculated at P / P0 = 0.99 using the BJH method (Barrett, E. P.; Joyner, L. G.; Halenda, P. P., “The Determination of Pore Volume and Area Distributions in Porous Substances I Computations from Nitrogen Isotherms” Journal of the American Chemical Society 1c951, 73 (1), 373-380). Pore diameter was assigned from the maximum of the BJH pore size distribution. In various embodiments, the overall pore volume and the mean pore diameter are reduced by treating the PMSNs with the SBS. Without being held to theory, it is believed that SBS modifies the interior surface of the pores as well as the outer surface of the NPs. In various embodiments it was determined that the radius of gyration of PEG5k is comparable to the pore diameter, which it is believed indicates, without being held to theory, that these molecules are end-grafted on the outer surface of the NPs. Assuming these molecules are end-grafted on the outer surface of the NPs, the grafting density of PEG5k chains on the outer surface of the PMSNs was estimated. This calculation uses the mass (or number) of PEG5kpolymers per PMSN and an estimate of the fraction of the outer surface of the PMSNs not occupied by pore openings. The estimation assumes that the NPs possess a perfect spherical shape and the mesopores are straight cylinders that penetrate the volume. This analysis leads to the following equation: ఘఉே ௗ×^ షమర ^^ =ಲೇ^ (2) where σ ρ represents the density of silica (1.85 - 2.20 g / cm3),38-39β is the weight ratio of mPEG5k in the sample obtained by TGA in units of mg / g, NAV is the Avogadro constant, d (in nm) is the average Attorney Docket No.: 4637-0019WO01 diameter measured by TEM, M is the molecular weight of the mPEG5k (5000 g / mol) and Vp is the pore volume obtained by nitrogen adsorption / desorption analysis using the BJH method (0.96 cm3 / g). The number 10-24converts unit of m2into nm2. A more detailed derivation of this equation is provided in Example 12. Because the silica density ρ can vary between 1.85 and 2.20 g / cm³, an estimate of between 0.52-0.84 chains / nm2of mPEG5k silane was used. This level of PEG5k grafting density generates polymer brushes at the particle surface. Lanthanide Element (Ln3+) Loading of the PEGylated MSNs. In order to transform the PMSNs-Zwi-mPEG5k NPs into useful reporters for mass cytometry, they have to be labeled with Ln3+ions. In various embodiments, metal ions we added as follow. We began with TbCl3·6H2O (10 mg / mL of Tb3+). Tb has only one naturally occurring stable isotope (159Tb with 100% abundance). This facilitates detection by inductively coupled plasma mass spectrometry (ICP-MS). The maximum loading content of Tb3+in PMSNs-Zwi-mPEG5kdetermined by ICP-MS was ca.19.6 mg / g, equivalent to 0.123 mmol / g. The particle number in solution was determined with a ZetaView Nanoparticle Tracking Analyzer (NTA). Combining the Tb content with the concentration of 1.0 × 1015NPs per gram obtained by NTA, the maximum Tb loading corresponded to ca.7.4 × 104Tb3+ / NP. From a mass cytometry perspective this is a useful value However, to serve as effective mass tags for mass cytometry, a number of other criteria have to be met. These include the ability to load different metal isotopes, stability against ion loss upon storage or application, surface functionalization for attachment of antibodies, and surface modification to minimize or prevent non-specific binding (NSB) to cells, which are provided for herein in various embodiments. In order to test the versatility of the various PMSNs-Zwi-mPEG5kNPs disclosed herein for loading various types of lanthanide ions, a mixture of metal salts comprising LaCl3·7H2O, CeCl3·7H2O, PrCl3·6H2O, NdCl3·6H2O, SmCl3·6H2O, EuCl3·6H2O, GdCl3·6H2O, TbCl3·6H2O, HoCl3·6H2O, Er(NO3)3·5H2O, TmCl3·6H2O, Yb(NO3)3·5H2O, LuCl3·6H2O (1 mg / mL of each metal ion) was mixed with nanoparticles (0.5 mg / mL) and stirred at room temperature for 24 h. Note that a large excess of the Ln3+ions was added. The loaded content of each lanthanide in the NPs was measured by ICP-MS, and ICP-MS analysis of the supernatant after NP isolation indicated that only about 0.1% of the Ln3+ions were taken up by the NPs. The data in FIG.9 Attorney Docket No.: 4637-0019WO01 shows that in various embodiments the PMSNs-Zwi-mPEG5k NPs provided herein can bind Ln3+ions across the lanthanide series and show some selectivity for the heavier lanthanides, particularly Yb3+and Lu3+. Lanthanide Ion Leaching Experiments. MSNs intended for drug delivery are designed to act as drug carriers but also to release loaded molecules at the target site, design goals counter to the needs of MC applications. For MC applications the MSNs must avoid ion loss upon storage and / or application. The stability of various embodiments of the metal-loaded MSNs in buffers was tested using Tb. The leaching of Tb ions from Tb@PMSNs-Zwi-mPEG5kin various buffers over a range of pH values was monitored by ICP-MS. The buffers ranged from 0.1 M Na2CO3 / NaHCO3 (pH 10.6), 0.1 M MES buffer (pH 5.5), 0.02 M Bis-tris buffer (pH 6.5), 0.01 M HEPES buffer (pH 7.4), H2O and 1× PBS buffer (pH 7.4). Individual plots of the time profile of Tb3+release are collected in FIGs.10A and 10B. Over a period of 4 days, there was no obvious detectable ion loss in H2O (~ 1.4%) and in 1× PBS buffer (~ 0.2%). However, for the case in 0.01 M HEPES buffer at pH 7.4, Tb ions leaked slowly from the NPs and the leakage reached about 13% after 4 days. For samples in 0.02 M Bis-tris buffer at pH 6.5 and 0.1 M MES buffer at pH 5.5, the losses of Tb ions were more substantial, amounting to 45% and 46%, respectively. Moreover, nearly 100% loss of Tb ion was found when Tb@PMSNs-Zwi-mPEG5k NPs were dispersed in 0.1 M Na2CO3 / NaHCO3 at pH 10.6. Various approaches to precipitation of the Tb inside the pores were performed to reduce the leaching, but several difficulties were presented. For example, preparation of colloidal hydrogels with europium (Eu) ions confined to the core are “insoluble” EuF3 NPs. However these microgels still leached Eu3+ions into the continuous medium. Experiments with Tb3+, with the approach of transforming TbF3 (Ksp(TbF3) ~ 10−19) to the much less soluble TbPO4 (Ksp(TbPO4) ~ 10−27), however, these attempts to precipitate TbPO4 inside the hydrogels by exposing them to several sources of inorganic phosphate led to precipitation outside of the hydrogels. It was discovered, however, that in various embodiments of the present disclosures that the TbF3-loaded hydrogels were stable in PBS buffer, and dispersing the microgels in PBS buffer rendered them stable to leaching and led to the formation of TbPO4nanocrystals in the interior. FIG.10A shows that Tb@PMSNs-Zwi-mPEG5k NPs are stable to leaching in PBS buffer. Referring to FIG.10B, the Attorney Docket No.: 4637-0019WO01 stability against leaching to Tb@PMSNs-Zwi-mPEG5k NPs pretreated with PBS was also examined and is was discovered that pretreatment also substantially reduced Tb leaching loss. Referring to FIG.11A, samples of Tb@PMSNs-Zwi-mPEG5kNPs were suspended in 1× PBS buffer, aged for various times (20 min, 40 min, 60 min, and 120 min), and then washed three times with H2O by sedimentation-redispersion (45,000 × g, 45 min). A sample was then transferred to 0.1 M MES buffer (pH 5.5) to investigate leakage. As displayed in FIG. 11A, regardless of the pretreatment time, leakage of Tb ions was effectively suppressed to less than 1%. Surprisingly, longer pretreatment times in 1× PBS led to somewhat greater loss of Tb ions. Accordingly, in various embodiments, pretreatment times of about 20 min, about 15 to 25 min, and less than about 30 min are preferred. The leakage analysis of Tb@PMSNs-Zwi-mPEG5k NPs in different buffer solutions was repeated after pretreatment. Compared to the leakage observed in Tb@PMSNs-Zwi-mPEG5kNPs without any pretreatment (FIGs.10A and 10B), there was little detectable Tb loss in the pretreated NPs over a four-day period (FIG.11B), except for 0.1 M Na2CO3 / NaHCO3 (pH 10.6) which still showed 13% leaching (FIG.10B). It is believed, without being held to theory, that the pretreated sample ion loss was due to the degradation of mesoporous silica nanoparticle frameworks under alkaline conditions, and this degradation mechanism was supported by TEM analyses (FIGs. 10C-10D). Thus, in various embodiments, provided are pretreatment methods that allow for employing various buffer solutions in mass cytometry studies with the functionalized MSNs provided herein, such as for example, Tb@PMSNs-Zwi-mPEG5k NPs. Determination of mPEG5k Grafting Density for Reduced or Minimized NSB. In various aspects and embodiments, the mPEG5k grafting density was adjusted by varying the NP-to-mPEG5k w / w ratio (NP / PEG) during the mPEG5k attachment step, and the extent of PEG grafting was quantified by TGA. The characteristics of the various PMSNs-Zwi-mPEG5k NPs synthesized in these examples are summarized in Table 3 below. Table 3. Physiochemical Characterization of series of PMSN-Zwi-mPEG5k NPs. NP / PEG w / w dTEM(nm) Coefficient of Variation dh PDI (CV) (nm) Attorney Docket No.: 4637-0019WO01 10:1047.7 ± 2.85.8% 83.3 0.14 10:20 47.5 ± 2.6 5.6% 84.8 0.13 10:4047.9 ± 3.16.5% 81.9 0.11 10:6048.2 ± 2.45.0% 82.0 0.15 10:8047.6 ± 2.65.5% 83.9 0.12 Table 4. Summary o af grafting densities of the mPEG5klayer and the parameters used to determine their conformation . Weight NP / PEG fting Density RFdDePEG Gra LfpercentagebConformation w / wc(^, chains / nm2) (nm) (nm) (nm) (wt%) of mPEG5ka10:10 6.5 (7.2) 0.27 (0.29) 5.8 2.1 11.3 Brush 10:20 8.4 (9.1) 0.34 (0.37) 5.8 1.9 12.3 Dense Brush 10:40 12.6 (13.3) 0.52 (0.54) 5.8 1.5 13.9 Dense Brush 10:60 11.4 (12.1) 0.47 (0.50) 5.8 1.6 13.6 Dense Brush 10:80 10.5 (10.9) 0.43 (0.45) 5.8 1.7 13.2 Dense BrushaThe prediction of PEG conformation was based on the model of Alexander de Gennes (“Conformations of Polymers Attached to an Interface”, Macromolecules 1980, 13 (5), 1069-b1075). Calculated from the mass loss determined by TGA. Duplicate experiments, the values from thecsecond set of measurements are given in parentheses.dCalculate F3d / 5with eq (2) R = αN , where α is the monomer length (0.35 nm for PEG)44, N is the number of PEG repeating unit (108 for mPEG5ke); Calculated mean spacing 2 between PEG anchor points, D = 2(1 / ^π)1 / 2, where ^ is the graftingfdensity (chains per nm ); Calculated brush height, L = N(α5 / 3) / D2 / 3The weight percent of mPEG5k in each sample was calculated from the mass loss obtained by TGA (FIG.12), summarized in Table 4. Referring to FIG.12A, the TGA curves reveal a noticeable pattern in the weight loss of mPEG5kas its addition increases. Referring to FIG.12B, the weight loss initially ascends to a peak value before gradually declining, aligning with the determined trend in grafting density. Notably, at an NP / PEG w / w ratio of 10:40, both the weight loss percentage and grafting density of mPEG5kreach their maximum values. Without being held to theory, using an assumption that PMSN nanoparticles can for the current purposes of surface Attorney Docket No.: 4637-0019WO01 area calculation be viewed as uniform spheres with dTEM= 45 nm, and the mesopores are analogous to straight cylinders, the surface area of the PMSNs was calculated. This value was combined with the measured loss of mPEG5kand the number of NPs in solution to calculate the grafting density of mPEG5k (^, chains per nm2) (Table 4). The conformation of end-grafted-PEG was determined according to the Alexander-de Gennes’ model (see, “Conformations of Polymers Attached to an Interface”, Macromolecules 1980, 13 (5), 1069-1075), using the Flory radius (RF) of PEG5k, the distance between adjacent PEG chain anchor points (D), and the length / thickness of the grafted PEG layer (L). Based on these parameters there are two main conformations that PEG chain can acquire, “mushroom” or “brush”. When D is greater than RF (RF / D < 1), the PEG chains on the surface of nanoparticles are highly flexible and cannot be completely extended, resulting in a mushroom conformation with a relatively thin PEG layer. Upon increasing the grafting density (RF / D > 1), the dense PEG chains generate elevated osmotic pressure within the medium, inducing the PEG chains to extend outward, thereby adopting a brush-like arrangement with a thicker layer. When the PEG layer length further exceeds RF by a factor of at least 2 (L / RF > 2), a distinct brush conformation is defined as a dense brush regime (see, e.g., Damodaran, V. B.; Fee, C. J.; Ruckh, T.; Popat, K. C., “Conformational Studies of Covalently Grafted Poly(ethylene glycol) on Modified Solid Matrices Using X-ray Photoelectron Spectroscopy”, Langmuir 2010, 26 (10), 7299-7306); an example is illustrated in FIG.12D. Following this model, as the mPEG5k grafting density increased from 0.27 to 0.54 chains per nm², various embodiments of the MSNs provided herein exhibited a brush- like or densely packed brush-like conformation. The thickness (L) of the PEG layer ranged from 11 to 14 nm, which was comparable to the value of Lh = 17 nm calculated (eq (1)) from the mPEG5k contribution to the hydrodynamic radius. These values are consistent with the PEG chains being in the form of a dense brush on the NP surface. In various embodiments, provided are compositions and methods to reduce and / or minimize NSB of PEGylated PMSNs by selection of the PEG surface coverage. Protein adsorption behavior was studied by incubating nanoparticles (5 mg / mL in 1× PBS) with human serum albumin solution (HSA, 1 mg / mL in 1× PBS) at 37 °C for 2 h (Thermomixer, 500 rpm). The weight percentage (wt%) of adsorbed HSA on NPs was assessed using the Pierce™ BCA protein assay (FIG.13A). As shown in FIG.12C, all of the PEGylated PMSNs significantly reduced Attorney Docket No.: 4637-0019WO01 HSA adsorption compared to bare MSNs and the PMSNs-Zwi sample. The PMSN-Zwi-mPEG5k NPs (10:40) sample exhibited the lowest serum albumin adsorption among the PEGylated samples of this example. Without being held to theory, is it believed that the densest brush-like conformation was most effective at inhibiting non-specific protein adsorption. Antibody Conjugation to PMSNs-Zwi-PEG5k-N3 NPs. In order to test the ability of NP-Ab conjugates of the present disclosures to act as reporters for mass cytometry analysis, the bioconjugation efficacy of Abs and NPs was examined and various conjugation conditions determined. NP-Ab conjugation utilized a DBCO-azide copper- free click chemistry cycloaddition reaction. Azide-containing PMSNs (PMSNs-Zwi-PEG5k-N3) were synthesized and subsequently conjugated to DBCO-modified Abs under various conjugation conditions. To test whether bioconjugation conditions preserved antigen recognition, an anti- biotin Ab was employed as a model, with biotin-Cy5 as the antigen target (FIG.14). Antigen recognition will give rise to a characteristic peak at λ = 649 nm under UV-vis detection. As a control, parallel experiments were carried out with mouse IgG2a, κ as an isotype control. PMSNs-Zwi NPs was reacted with N3-PEG5k-silane to prepare PMSNs-Zwi-PEG5k-N3 NPs under reaction conditions similar to those used to prepare PMSNs-Zwi-mPEG5k. FIG.15 shows the TEM image of PMSNs-Zwi-PEG5k-N3 NPs. The characteristic dimensions of these NPs were dTEM= 49.1 ± 3.1 nm and dh = 79.9 nm (PDI = 0.095), respectively, similar to those of PMSNs- Zwi-mPEG5kNPs (see, e.g., FIGs.2C and 2D). These PMSNs-Zwi- PEG5k-N3NPs could also be well-redispersed in 1× PBS even after drying, showing good stability when stored in PBS at 4 °C for up to 7 days (FIG.15C). The presence of active azide functional groups on PMSNs-Zwi- PEG5k-N3 NPswas determined by reacting them with DBCO-Cy5 at room temperature for 12 h. After purification, the UV-vis absorbance of PMSNs-Zwi-PEG5k-N3 NPs in 1× PBS was measured and the characteristic absorbance peak at 649 nm attributed to Cy5 group could be clearly observed (FIG.15D). The number of azide groups per PMSNs-Zwi-PEG5k-N3 NP was calculated to be ~ 630. The purified anti-biotin Abs was reacted with the mouse IgG2a, κ isotype control Abs with 10-fold molar excess of DBCO-PEG4-NHS to introduce DBCO functional groups to the Abs. After the reaction, the purified DBCO-Ab solutions were measured by UV-vis. The spectra show Attorney Docket No.: 4637-0019WO01 two characteristic absorbance peaks of DBCO at 309 nm and Ab at 280 nm (FIG.16A). The number of DBCO groups per Ab was calculated as approximately 4 using eq (S1). The reaction of DBCO-Abs with PMSNs-Zwi-PEG5k-N3NPs was carried out at Ab-to-NP ratios of 22:1 or 50:1, and with different conjugation conditions of (a) 37 °C, 1 h, (b) 37 °C, 4 h, (c) room temperature (RT, 25 ^C), 4 h, and (d) RT, 12 h. The excess unconjugated Abs were removed by multiple washes in a PALL spin filter (300 kDa, 2,080 × g for 20 min). FIG.16B shows the DLS results of the purified PMSNs-Zwi-PEG5k-anti-biotin Ab conjugates obtained after a 4-hour incubation at RT. An approximate 10 nm increase in the hydrodynamic diameter was observed when comparing the NP-anti-biotin Ab conjugates to the PMSNs-Zwi-PEG5k-N3 NPs. This increase closely matches the size of the anti-biotin antibody (dh = 10.7 nm) as determined by DLS. The number of Ab per NP was determined by a Micro™ BCA protein assay with the calibration curve plotted in FIG.13B. During the bioconjugation process at RT for 4 h, for example, approximately 9 anti-biotin Abs or 6 isotype control Abs were attached to each NP with a 22:1 feed ratio, while a 50:1 feed ratio yielded ca.13 anti-biotin Abs per NP or 8 isotype control Abs per NP. It was determined that for each bioconjugation condition the higher feed ratio of Ab to NP led to higher amounts of Ab attached per NP. To determine if the reactivity of anti-biotin Abs was maintained after conjugation and purification, NP-anti-biotin Ab conjugates were incubated with a 4-fold excess of Cy5 biotin molecules in a ThermoMixer (Eppendorf, 500 rpm) at RT for 2 h. After removing the unbound Cy5 biotin through five cycles of spin filtration (Amicon, Ultra-0.5, 100 kDa, 4,100 × g for 20 min, washed with H2O), UV-vis spectroscopy measurements were performed on eight different NP-anti-biotin Ab-Cy5 conjugates. The isotype control Ab-conjugated NPs obtained from various conjugation conditions were used as negative controls, being put through the same process of incubation with Cy5 biotin, spin filtration, and UV-vis detection. A characteristic absorbance peak at 649 nm was observed in the case of NP-anti-biotin Ab-Cy5 conjugates, whereas no such peak could be observed for the NP-control Ab-Cy5 conjugates (FIG. 16C, see inset). This phenomenon remained constant under all 8 different bioconjugation conditions, indicating that the anti-biotin Abs are still functional and capable of binding their target after the conjugation and purification procedures. The proportion of active Abs was quantified by counting the amount of Cy5 per Ab, and was used to evaluate the various conjugation conditions embodiments ability to preserve Ab Attorney Docket No.: 4637-0019WO01 functionality. The UV-vis spectra of the precursor NP-anti-biotin Ab conjugates was normalized to that of the NP-anti-biotin Ab-Cy5 conjugates in the wavelength range of 400 to 550 nm, where the dye Cy5 does not absorb. The corrected baseline is shown as a dashed line in FIG.16C. The difference between this corrected baseline and the absorbance peak at 649 nm was taken as the 5 measure of the amount of Cy5 biotin molecules captured by the anti-biotin Abs. The number of Cy5 per NP was calculated using eq (S2). Consequently, the Cy5-to-Ab ratio was determined by comparing the quantity of Cy5 per NP to the quantity of Ab per NP. A similar number of Cy5 biotin molecules per Ab was found across all conjugation conditions of this example when the Ab-to-NP feed ratio was set at 22:1 (FIG.16D). In contrast, with a 50:1 10 ratio, the number of Cy5 per Ab was higher. It is important to note that each IgG Ab has two identical antigen-binding sites (in our system, specific for biotin). Nevertheless, none of the reaction conditions in the present example allowed for the detection of such a high level of biotinylated-Cy5 capture. It was determined, however, that values of Cy5 per Ab greater than 1.0 were obtained and that higher Ab-to-NP feed ratio and gentler bioconjugation conditions favor 15 Abs with greater retention of functionality. The inventions and there various aspects and embodiments will be illustrated in more detail with reference to the following Examples, but it should be understood that the present inventions, aspects and embodiments are not deemed to be limited thereto. 20 EXAMPLES Example 1 -Instrumentation. Transmission Electron Microscopy (TEM): The NP samples were dispersed in H2O and drop-cast on Formvar / Carbon 200 mesh grids. All TEM images were obtained on a Hitachi HT7700 TEM instrument operating at 80 kV. Particle sizes were measured using Image-J, and 25 size distribution histograms were plotted using Origin. Dynamic Light Scattering (DLS) Measurements: DLS measurements were performed at 25 °C on Malvern Zetasizer Nano ZS instrument. Aqueous samples were placed in ZEN0040 disposable plastic micro cuvettes. Samples were dispersed in water or 1× PBS buffer at a concentration of ~ 0.1 mg / mL. Each size measurement was performed at a scattering angle of Attorney Docket No.: 4637-0019WO01 173°. Z-average hydrodynamic diameters (dh), polydispersities (PDI) and intensity distribution plots were obtained using software embedded in the instrument. Fourier-Transform Infrared (FTIR) Spectroscopy: All FTIR spectra were collected using a PerkinElmer Spectrum Two ATR-FTIR with a diamond crystal polarization accessory.64 scans were performed on each sample at 4.0 cm-1resolution. Thermogravimetric Analysis (TGA): Quantitative determination of functional groups and polymers bound to the MSN surface was conducted on SDT Q600 thermogravimetric analyzer in nitrogen at a flow rate of 100 mL / min. The temperature was ramped to 100 °C, where it was held constant for 30 min to ensure the desorption of adsorbed water. Finally, the temperature was ramped to 800 °C at 10 °C / min. Nitrogen adsorption / desorption isotherms. Nitrogen adsorption / desorption isotherms were measured on a Quantachrome Instruments Autosorb-iQ (Boynton Beach, Florida USA) with the extra-high pure gases. The samples were degassed in a gradient way for 12 h in total. The surface areas were determined from adsorption isotherms by the Brunauer-Emmett-Teller (BET) method (see, Konry, T.; Smolina, I.; Yarmush, J. M.; Irimia, D.; Yarmush, M. L., “Ultrasensitive Detection of Low-Abundance Surface-Marker Protein Using Isothermal Rolling Circle Amplification in a Microfluidic Nanoliter Platform”, Small 2011, 7 (3), 395-400), while pore size and pore volume were calculated from desorption branches of isotherms by the Barrett-Joyner- Halenda (BJH) method (see, Behbehani, G. K., “Applications of mass cytometry in clinical medicine: the promise and perils of clinical CyTOF:, Clinics in laboratory medicine 2017, 37 (4), 945-964). Nuclear Magnetic Resonance (NMR) Spectroscopy:1H NMR measurements were carried out on either a 400 MHz Agilent DD2 NMR Spectrometer or a 600 MHz Agilent DD2 NMR Spectrometer. Inductively Coupled Plasma Mass Spectrometry (ICP-MS): All measurements were performed using a Thermo Scientific iCAP Q ICP-MS spectrometer. The 2 v / v% HNO3solution in ultrapure water was prepared from high purity HNO3. The calibration standards were prepared from the standard solution in 2 v / v% HNO3 with a series concentration of 0.1, 1, 10, 20, and 40 ppb. An internal standard of ca.20 ppb was used. All samples were diluted 50, 100, and 500 times, respectively to get the optimal concentration lying among the calibration concentrations. Attorney Docket No.: 4637-0019WO01 Each measurement was conducted in triplicate, and the final ppb values of interest elements were calculated as the average from all measurements at different dilutions. ZetaView Nanoparticle Tracking Analyzer (NTA): A dilute sample of 100 nm polystyrene microbeads (NanosphereTM, Thermo Fisher Scientific) was used as a standard solution to calibrate the instrument. The sample solution (1.5-2 mL) with a known concentration (mg / mL) was injected into the instrument for measurement. The NP concentration determined is given in units of particles / mL, and with the known mass concentrations, these values could be converted to particles / gram. Ultraviolet-visible (UV-vis) Spectrophotometer: UV-vis measurements were performed on a BioTek Epoch 2 microplate spectrophotometer. All sample dispersions were placed in either BrandTech™ BRAND™ disposable cuvettes with a 1 cm path length or Thermo Scientific Pierce™ 96-well plates. Example 2 – Materials Tetraethyl orthosilicate (TEOS), triethanolamine (TEA), triethylamine, hexadecyl trimethyl ammonium chloride (CTAC, 25 wt%), (N,N-dimethylaminopropyl)trimethoxysilane (DMASi), 1,3-propane sultone, sodium azide, dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, ≥ 90%), albumin from human serum (HSA, protease free, ≥ 96%), metal salts with purities of ≥ 99.99% (trace metal basis), including lanthanum(III) chloride heptahydrate (LaCl3·7H2O), cerium(III) chloride heptahydrate (CeCl3·7H2O), praseodymium chloride hexahydrate (PrCl3·6H2O), neodymium(III) chloride hexahydrate (NdCl3·6H2O), samarium (III) chloride hexahydrate (SmCl3·6H2O), europium(III) chloride hexahydrate (EuCl3·6H2O), gadolinium (III) chloride hexahydrate (GdCl3·6H2O), terbium(III) chloride hexahydrate (TbCl3·6H2O), holmium(III) chloride hexahydrate (HoCl3·6H2O), erbium(III) nitrate pentahydrate (Er(NO3)3·5H2O), thulium(III) chloride hexahydrate (TmCl3·6H2O), ytterbium (III) nitrate hydrate (Yb(NO3)3·5H2O), lutetium(III) chloride hexahydrate (LuCl3·6H2O), single- element standard solutions (i.e., Ho, Tb and Ta) and multi-element standard solution for inductively coupled plasma mass spectrometry (ICP-MS) calibration were purchased from Sigma-Aldrich (CA). Silane-PEG5k-methoxy (M 5000 g / mol) was purchased from JenKem Technology (USA). Silane-PEG5k-azide was purchased from Biopharma PEG (Watertown, MA, USA). 3-Methoxy(polyethyleneoxy)6-9-propyltrimethoxysilane (PEG6-9 silane, 459-591 g / mol) was purchased from Gelest (Morrisville, PA, USA). Phosphate-buffered saline (1× PBS solution Attorney Docket No.: 4637-0019WO01 which is 0.137M NaCl, 0.0027M KCl, and 0.0119M phosphates and pH 7.4 , Fisher BioReagents), high purity nitric acid (trace metal grade, 67-70%, Optima™) used for ICP-MS, Pierce™ BCA Protein Assay Kit and Micro™ BCA Protein Assay Kit were purchased from Fisher Scientific. Cy5 biotin was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Purified anti-biotin antibody (Clone, 1D4-C5) and isotype control antibody (purified mouse IgG2a, κ, clone MOPC- 173) were purchased from BioLegend. Antibodies were purified by spin filtration (Amicon, Ultra- 0.5, 10 kDa) with 1× PBS to remove sodium azide before use. All other chemicals were used as received without further purification. Example 3 - Synthesis of PEG6-9-modified Mesoporous Silica Nanoparticle (PMSNs). The PMSNs were synthesized as follows. Hexadecyl trimethyl ammonium chloride (CTAC, 2 g) and triethanolamine (TEA, 0.8 g) were dissolved in water (18.2 MΩ, 20 mL) and magnetically stirred at room temperature for 1 h. The mixture reaction was heated to 95 °C and stirred for another 1 h. Tetraethyl orthosilicate (TEOS, 1.5 mL) was injected into the reaction using a syringe pump at a rate of 150 μL / min. After the TEOS injection, the mixture was stirred for 10 min before 3-[methoxy(polyethyleneoxy)6-9]-propyltrimethoxysilane (PEG6-9-silane, 650 μL) was injected into the solution at a rate of 65 μL / min. This step was followed by stirring at 95 °C for another 30 min. After the reaction mixture was cooled to room temperature, the resulting CTAC-containing PMSNs were collected by three cycles of sedimentation-redispersion (45,000 × g, 45 min) with ethanol. In order to remove the CTAC template, the NPs were dispersed in 50 mL of acidic ethanol solution (10 v / v% of concentrated HCl in ethanol) and refluxed at 85 °C for 12 h. This extraction was carried out twice to ensure that CTAC was completely removed. Finally, the PMSNs were collected by three cycles of sedimentation-redispersion (45,000 × g, 45 min) with ethanol, then dried under vacuum oven overnight. Example 4 - Synthesis of Zwitterion-modified Mesoporous Silica Nanoparticle (PMSNs- Zwi). For zwitterionic sulfobetaine silane (SBS) synthesis, a round-bottom flask (25 mL) with a magnetic stirrer was sealed with a rubber septum and purged with N2 for 30 min. (N,N- dimethylaminopropyl)trimethoxysilane (DMASi, 2.07 g, 10 mmol), 1,3-propane sultone (1.34 g, 11 mmol) and anhydrous acetone (10 mL) were added under N2 into the reaction flask with a syringe. The reaction was stirred vigorously at room temperature for 6 h. The white precipitate Attorney Docket No.: 4637-0019WO01 was centrifuged (4,000 rpm, 10 min) and washed 3 times with anhydrous acetone in order to remove the unreacted reagents. The final product was dried at 30 °C under vacuum for 24 h and stored under N2. The yields were between 60 and 75 %.1H NMR and13C NMR spectra of the products were obtained with a 400 MHz Agilent DD2 Spectrometer and presented in FIGs. 3B and 3C. A sample of PMSNs (50 mg) was redispersed in H2O (4 mL) under sonication, resulting in a transparent light blue solution. The solution was purged with N2 for at least 15 min. Zwitterionic sulfobetaine silane (50 mg) was dissolved in H2O (1 mL) prior to dropwise addition into the PMSN solution. The pH was adjusted to ∼ 9 using 28 % aqueous ammonia. The mixture was stirred at 80 °C for 24 h. The resulting nanoparticles were washed by three cycles of sedimentation-redispersion (45,000 × g, 45 min) with excess H2O in each cycle and freeze-dried. Example 5 - Synthesis of PEG5k silane-modified Mesoporous Silica Nanoparticle (PMSNs- Zwi-PEG5k). PEGylated PMSN-Zwi NPs with different functional groups were prepared using silane- PEG5k-methoxy (mPEG5k) or silane-PEG5k-azide (N3-PEG5k) under similar conditions. Typically, PMSNs-Zwi (10 mg) was added into a two-neck round-bottom flask (25 mL) and suspended in anhydrous toluene (5 mL). The resulting suspension was purged with N2for 15 min and a mPEG5ksolution in 1 mL anhydrous toluene was added dropwise into the suspension. The reaction was stirred at 110 °C for 24 h. After cooling, the nanoparticles were collected through centrifugation (45,000 × g, 45 min) and washed three times with ethanol to remove the unreacted mPEG5k silane. The final PMSNs-Zwi-mPEG5kwere suspended in water and lyophilized to obtain a free-flowing powder. By adjusting the amount of mPEG5k from 10 mg to 80 mg under the same conditions, samples with different mPEG5k grafting densities could be obtained. Additionally, PEGylated MSNs with azido functional groups (PMSNs-Zwi-PEG5k-N3) were prepared under the same conditions by using N3-PEG5k silane with the mass ratio of nanoparticles to PEG silane of 10:40. Example 6 - Lanthanide element loading and leaching analysis. In a typical loading experiment, 10 mg of the PMSNs-Zwi-mPEG5kNPs were dispersed into H2O (2 mL) under sonication. TbCl3·6H2O (47 mg, 10 mg / mL of Tb) was added into the suspension and the mixture was stirred at room temperature for 12 h. Tb-loaded PMSNs-Zwi- mPEG5k(Tb@PMSNs-Zwi-mPEG5k) NPs were obtained by centrifugation (45,000 × g, 45 min) and washed with water twice. 20 mL of 1× PBS buffer was added after the last run of Attorney Docket No.: 4637-0019WO01 centrifugation in order to precipitate Tb3+as TbPO4 inside the pores. The Tb-containing nanoparticles were immersed in 1× PBS buffer at room temperature without stirring for 20 min, 40 min, 60 min or 120 min. Then the nanoparticles were washed with H2O by three cycles of sedimentation-redispersion (45,000 × g, 45 min). After freeze-drying, the Tb@PMSNs-Zwi- mPEG5k NPs were dispersed in 2 v / v% HNO3 and the amount of Tb element in nanoparticles was determined by ICP-MS analysis. For multi-element loading, a sample of PMSNs-Zwi-mPEG5k in water (2.5 mg, 0.5 mg / mL, 5.0 mL) was prepared. To this solution was added sequentially a series of lanthanide salts in amounts that would lead to a final concentration of 1 mg / mL of each. Pr3+, Er3+and Yb3+were added as nitrate salts, and La3+, Ce3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Ho3+, Tm3+, and Lu3+were added as chloride salts. This mixture was stirred at room temperature for 24 h. Leaching of Ln ions from lanthanide-loaded NPs was tested using Tb@PMSNs-Zwi- mPEG5k. For each measurement, an NP sample (3 mg) was dispersed in a buffer solution (5 mL) and stirred vigorously at room temperature. Different buffers including 0.1 M sodium carbonate / bicarbonate buffer (Na2CO3 / NaHCO3, pH 10.6), 0.1 M MES buffer (pH 5.5), 0.02 M Bis-tris buffer (pH 6.5), 0.01 M HEPES buffer (pH 7.4), H2O and 1× PBS buffer were utilized for these leaching tests. Then aliquots (0.5 mL) were taken and transferred into 1.5 mL centrifuge tubes from time-to-time over 7 days. Each sample was centrifuged at 45,000 × g for 45 min. After centrifugation, the supernatant was diluted 50 times with 2 v / v% HNO3solution for ICP-MS analysis. Example 7 - Serum Protein Adsorption Analysis. Protein adsorption experiments were performed at 37 °C for 2 h using protease free human serum albumin (HSA). A nanoparticle sample (1.000 ± 0.040 mg, weighed on a microbalance) was redispersed in 1× PBS buffer (200 μL) under sonication. Then an HSA solution in 1× PBS buffer (200 μL, 1 mg / mL) was added. The mixture was incubated in an Eppendorf ThermoMixer at 37 °C at a shaking rate of 500 rpm for 2 h. After incubation, loosely bound proteins were removed using an Amicon Ultra-0.5 spin filter (100 kDa, 10,000 rpm, 10 min) and washed 5 times with fresh 1× PBS buffer. The amount of adsorbed HSA proteins was determined using a Pierce™ BCA assay (Thermo Fisher Scientific) at λ = 562 nm. The supernatant after each run of centrifugation was collected and analyzed by UV-vis in order to make sure that all loosely bound proteins were removed. Attorney Docket No.: 4637-0019WO01 Example 8 - Conjugation of Antibodies to PMSNs-Zwi-PEG5k-N3 NPs; Preparation of DBCO-functionalized antibody (DBCO-Ab). Two types of Abs were used for bioconjugation following the same protocol: an anti- biotin Ab and a control Ab. As an example, Ab stock solution (100 μL, 1.6 mg / mL in 1× PBS buffer, pH 7.4) was incubated with DBCO-PEG4-NHS stock solution (1.1 μL, 10 mM in anhydrous DMSO, 10 molar equivalents) at room temperature for 1 h. Unreacted DBCO-PEG4- NHS was removed using spin filtration (Amicon, Ultra-0.5, 10 kDa) at 12,000 × g for 10 min and washed three times with 1× PBS buffer. The DBCO-Ab solution was then diluted with 1× PBS buffer (pH 7.4) to a total volume of 200 μL. The Ab concentration and the number of DBCO groups per Ab were determined by UV-vis measurements. The number of DBCO groups per Ab was calculated according to the following equation: ಲ(యబవ ^^) ^^^^^^^^ ^^^^^^ ^^^^ =^(ವಳ^ೀ)ಲ(మ^బ ^^)షభ.బ^వ ×ಲ(యబవ ^^) (S1) where A (309 nm) and A 309 nm and λ = 280 nm. ɛ (DBCO) and ɛ (Ab) are the molar extinction coefficients of DBCO (12,000 M-1·cm-1) and Ab (210,000 M-1·cm-1).1.089 is the DBCO correction factor at 280 nm. Example 9 - Bioconjugation of DBCO-Abs with PMSNs-Zwi-PEG5k-N3 NPs (NP-Ab). For bioconjugation, PMSN-Zwi-PEG5k-N3 NPs were used and conjugated with DBCO- Ab via click chemistry. The conjugation reaction was performed under several different conditions, i.e., 37 °C for 1 h, 37 °C for 4 h, room temperature for 4 h or room temperature for 12 h. Different amounts of DBCO-Ab solution were mixed with NP solutions (5 mg / mL, 100 μL) to adjust the Ab-to-NP ratio to 22:1 or 50:1. In a typical reaction with an Ab-to-NP ratio of 50:1, DBCO-Ab in 1× PBS buffer (45 μL, 0.4 mg / mL) was mixed with PMSN-PEG5k-N3 NPs in 1× PBS buffer (100 μL, 5 mg / mL), where the concentration of DBCO-Ab in the resulting mixture was ~ 0.86 μM. The conjugation reaction was performed at 37 °C for 4 h with a ThermoMixer (Eppendorf, 500 rpm), then quenched by adding sodium azide (8 μL, 1 wt % in deionized water, room temperature, 1 h). The NP-Ab conjugate solution was spin filtered (PALL, 300 kDa) at 2,080 × g for 20 min and washed four times with 1× PBS buffer to remove unreacted DBCO-Abs and sodium azide. Finally, an additional PBS buffer (pH 7.4) was added to adjust the final volume to 100 μL. Then 15 μL of the purified conjugate solution was diluted 10 times for a Micro™ BCA assay to quantify the number of Ab per NP. Attorney Docket No.: 4637-0019WO01 Example 10 - Labeling NP-Ab with Cy5 Biotin and Quantification of Active Ab. Labeling NP-Ab with Cy5 Biotin (NP-Ab-Cy5). Based on the number of Ab per NP, a 4-fold excess of Cy5 biotin was mixed with the NP-Ab conjugate solution. In a typical reaction, NP-Ab solution (60 μL) was mixed with Cy5 biotin solution (0.85 μL, 0.2 mg / mL in H2O). The mixture was vortexed at room temperature for 2 h with a ThermoMixer (Eppendorf, 500 rpm). After the reaction, the mixture was purified by spin filtration (Amicon, Ultra-0.5, 100 kDa) at 4,100 × g for 15 min and washed five times with H2O. Additional H2O was added to reach a final volume of 60 μL. The number of Cy5 per NP was quantified from UV-vis spectra. Quantification of Cy5 Per Ab. Prior to incubation with Cy5 biotin, each NP-Ab solution was measured by UV-vis. The wavelength ranging from 400 nm to 550 nm of the NP-Ab spectrum was normalized to the corresponding NP-Ab-Cy5 spectrum using the least squares method. This normalized NP-Ab spectrum served as a model baseline. The absolute absorbance value of Cy5 (A649) was obtained by subtracting the absorbance value at λ = 649 nm in the normalized NP-Ab spectrum from the absorbance value at λ = 649 nm in the corresponding NP-Ab-Cy5 spectrum. The number of Cy5 groups per NP was calculated according to the following equation: ^^^^5 ^^^^^^ ^^^^ =^లరవ·ேಲೇ ିଷఌ(^௬ହ)·^ಿ^ಲ(ே^)× 10 (S2)where NAVis Avogadro’s - M1·cm-1), cNTA(NP) is the NP concentration (particles / mL) obtained from the NTA. The factor 10-3is used to convert units of mL into L. The number of Cy5 per Ab was calculated as the ratio of Cy5 per NP to Ab per NP. Example 11 – Characterization of Surface Modification by1H NMR The surface modification of the MSNs was also examined by1H NMR. FIG.8, compares the spectra of PMSNs, PMSNs-Zwi, and PMSNs-Zwi- in D2O. In the1H NMR spectrum of PMSNs, a peak related to the hydrogens of -CH2-CH2O- at 3.5-3.6 ppm is observed, and attributed to the PEG6-9 silane incorporated into the nanoparticles in the first synthesis step. After modification with SBS, PMSNs-Zwi retained the resonance signals corresponding to PEG units and closely resembled the SBS spectrum shown in FIG. 3. After introduction of the PEG5kpolymers, the signal area attributed to the hydrogen atoms of -CH2- CH2O- markedly increased, while the peak of SBS remained visible. These results clearly indicate the presence of both SBS and mPEG5k silane in the multi-functionalized nanoparticles. Attorney Docket No.: 4637-0019WO01 Example 12 - Derivation of the Equation for Estimating mPEG5k Grafting Density ఘఉே ௗ×^ షమ sாீ / ^^=ಲೇ^ ^మ^ெ[^ିഐೇ^(2) య ] where ^PEG / nm2 represents ρ represents the density of obtained by TGA in units of mg / g, NAVis Avogadro’s number, d (nm) is the average diameter measured from TEM images, M is the molecular weight of the mPEG5k (5000 g / mol) and Vp is the pore volume obtained by nitrogen adsorption / desorption analysis using BJH method (0.96 cm3 / g). The factor 10-24is used to convert units of m2into nm2. In order to determine the surface area available for PEG modification, one must subtract the void surface attributed to the mesoporous openings (Spore) from the overall surface area of the sphere (Ssph). Therefore, షమ s ఉఘ^ ^ாீ / ^^మ=ೞ^^ேಲೇ×^^ ெ(ௌೞ^^ିௌ^^^^) (S3) where Vsphis the volume of = 4 / 3π(d / 2)3, Ssph is the area a as = πd2. Therefore, eq (S3) could be converted to: s ఉఘே ×య షమ^ =ಲೇగௗ ×^^ ^ாீ / ^మ(S4) Spore represents the void diameter (dpore) is the mesopore size obtained from the BJH data. Consequently, Spore could be calculated by multiplying dporeand the number of pores per nanoparticle (npore), where nporeis equal to twice the base of each cylinder (2ncylinder): ^^ = ^^ ^^ ^ௗ^^^^ଶ = ௗ^^^^ ଶ^^^^ ^^^^^ The number of pore nanoparticle (Vpore ‧ ρVsph) by the cylinder volume (Vcylinder), where Vcylinder is estimated by using the diameter (d) of nanoparticle as the height: ^ ర ^ ^^^^×ఘ^ೞ^^^^^^^×ఘ× మ యగ( మ)యଶ^^^^^ఘௗ ^^^ ^^ Attorney Docket No.: 4637-0019WO01 Combining eq (S5) and eq (S6) and introducing these terms into eq (S4) gives eq (2). The estimated mPEG5kdensity on the outer surface of the mesoporous silica nanoparticles was calculated accordingly. Example 13 - Determination of mPEG5k Conformation Flory radius (RF), grafting distance (D) and thickness of mPEG5k layer (L) were determined using the following equations: RF = αN3 / 5(S7) D = 2(1 / ^π)1 / 2(S8) where α is the monomer (108 for mPEG5k used in this , per While the inventions have been described in detail and with reference to specific examples, aspects and embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS All references throughout this application, for example patent documents, including issued or granted patents or equivalents and patent application publications, and non-patent literature documents or other source material are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference. None is admitted to be prior art. When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups and classes that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. As used herein, “and / or” means that one, all, or any combination of items in a list separated by “and / or” are included in the list; for example “1, 2 and / or 3” is equivalent to “1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2, and 3”. Attorney Docket No.: 4637-0019WO01 Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of materials are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same material differently. It will be appreciated that methods, device elements, starting materials, and synthetic 5 methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials, and synthetic methods are intended to be included in this invention. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all 10 individual values included in the ranges given are intended to be included in the disclosure. It is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower 15 limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included. 20 As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the 25 claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition, in a description of a method, or in a description of elements of a device, is understood to encompass those compositions, methods, or devices consisting essentially of and consisting of the recited components or elements, optionally in addition to other components or elements. The invention illustratively described herein suitably may be practiced in the absence Attorney Docket No.: 4637-0019WO01 As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “the nanoparticle” includes reference to one or more nanoparticles and equivalents thereof known to those skilled in the art, 5 and so forth. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed 10 by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. 15

Claims

Attorney Docket No.: 4637-0019WO01 CLAIMS WHAT IS CLAIMED IS:

1. A composition comprising functionalized mesoporous silica nanoparticles (MSN), wherein the functionalized MSN comprises: mesoporous silica nanoparticles (MSN); at least one zwitterionic silane; and at least one polyethylene glycol (PEG) functionalized silane.

2. The composition of claim 1 wherein the at least one zwitterionic silane is a zwitterionic betaine group containing silane.

3. The composition of claim 1 wherein the at least one zwitterionic silane is a zwitterionic sulfobetaine silane.

4. The composition of 3 wherein the at least one polyethylene glycol (PEG) functionalized silane is a PEG5ksilane.

5. The composition of claim 1 wherein the functionalized MSN further comprises lanthanide ions.

6. The composition of claim 5 wherein the lanthanide ion is selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof.

7. The composition of claim 1wherein the functionalized MSN further comprises Tb ions.

8. The composition of claim 6 wherein the functionalized MSN comprises at least about 1 x 104lanthanide ions per functionalized MSN.

9. The composition of claim 6 wherein the functionalized MSN comprises at least about 5 × 104lanthanide ions per functionalized MSN.

10. The composition of claim 4 wherein the PEG5k-silane is an azide-PEG5k-silane.

11. The composition of claim 1 wherein the functionalized MSN is conjugated with dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs).Attorney Docket No.: 4637-0019WO01 12. The composition of claim 2 wherein the PEG5k-silane is an azide-PEG5k-silane conjugated with a dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs).

13. A mixture of functionalized MSNs, the mixture comprising a plurality of functionalized MSNs, each functionalized MSN comprising, a mesoporous silica nanoparticle (MSN); at least one zwitterionic silane attached to the MSN; at least one polyethylene glycol (PEG) functionalized silane attached to the MSN; and at least one lanthanide ion attached to the MSN.

14. The mixture of claim 13 wherein the at least one zwitterionic silane is a zwitterionic sulfobetaine silane.

15. The mixture of 13 wherein the at least one polyethylene glycol (PEG) functionalized silane is a PEG5ksilane.

16. The mixture of claim 15 wherein the PEG5k-silane is an azide-PEG5k-silane 17. The mixture of claim 13 wherein the lanthanide ion is selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof.

18. The mixture of claim 16 wherein the functionalized MSN comprises at least about 1 x 104lanthanide ions per functionalized MSN.

19. The mixture of claim 13 wherein at least a portion of the MSNs further comprise at least one antibody attached to at least one of the polyethylene glycol (PEG) functionalized silanes.

20. The mixture of claim 13 wherein the mixture is lyophilized.

21. A method for making a functionalized mesoporous silica nanoparticle reagent, comprising the steps of providing a mixture of functionalized mesoporous silica nanoparticles, each functionalized a mesoporous silica nanoparticle (MSN) comprising a mesoporous silica nanoparticle (MSN), at least one zwitterionic silane attached to the MSN, at least one polyethylene glycol (PEG) functionalized silane attached to the MSN, and at least one lanthanide ion attached to the MSN;Attorney Docket No.: 4637-0019WO01 contacting the mixture with a phosphate-buffered saline (PBS) having a pH in the range between about 7.3 to 7.5, a NaCl concentration of about 0.14M, a KCl concentration of about 0.003M, and a total phosphates concentration of about 0.02M, and reacting the PBS with the mixture of functionalized MSNs at a temperature in the range 5 between about 20 °C to about 25 °C for a time between about 10 minutes and about 40 minutes to produce the functionalized mesoporous silica nanoparticle reagent.

22. The method of claim 21 wherein the reaction time is between about 15 minutes and about 25 minutes.

23. The method of claim 21 wherein the at least one zwitterionic silane is a zwitterionic sulfobetaine silane.

24. The method of claim 21 wherein the at least one polyethylene glycol (PEG) functionalized silane is a PEG5ksilane.

25. The method of claim 21 wherein the PEG5k-silane is an azide-PEG5k-silane 26. The method of claim 21 wherein the lanthanide ion is selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof.

27. The method of claim 21 wherein the functionalized MSN comprises at least about 1 x 104lanthanide ions per functionalized MSN.

28. The method of claim 21 further comprising the step of lyophilizing the functionalized mesoporous silica nanoparticle reagent to produce a lyophilized functionalized mesoporous silica nanoparticle reagent.

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