Non-specific Recognition Research Articles

Poly(p-Phenyleneethynylene)s-Based Sensor Array for Diagnosis of Clinical Diseases.

Inspired by the mammalian taste and olfactory systems, array-based pattern recognition technology has demonstrated significant potential in discerning subtle differences between highly similar compounds and complex mixtures, owing to their unique parallel detection mechanism based on cross-reactive signals. While optical sensor array has been extensively employed in the field of chemical sensing, they encounter significant challenges in non-specific recognition of multiple analytes at low concentrations, particularly in rife environments with complex interferences. Poly(p-phenylene ethynylene)s (PPEs) offer substantial advantages in detecting multi-analytes at low concentrations, owing to its distinctive optical properties, including the "molecular wire" effect, fluorescence super-amplification and super-quenching. This is particularly promising for the parallel detection of ultra-low concentration multi-biomarkers in clinical diseases. As the continuous development of PPEs sensor array, more sensitive methods for rapid detection of clinical disease will be further developed. It will promote the further development of the field of early diagnosis of clinical diseases.

A novel carboxylesterase 2-targeted fluorescent probe with cholic acid as a recognition group for early diagnosis of drug- and environment-related liver diseases

Due to the detrimental effects of various harmful substances—such as carcinogens, drug toxicity, and environmental pollutants—on the liver, which can trigger or exacerbate conditions like hepatocellular carcinoma (HCC), drug-induced liver injury (DILI), and non-alcoholic fatty liver disease (NAFLD), accurate detection and monitoring of these diseases are crucial for effective treatment. Carboxylesterase 2 (CES2) is primarily found in the liver and, as a potential biomarker, its accurate detection can enhance the early diagnosis and treatment efficacy of liver diseases. Traditional fluorescence probes for CES2 detection suffer from non-specific recognition groups, leading to poor targeting specificity. To address this limitation, we propose a novel CES2-responsive fluorescent probe utilizing cholic acid (CA) as a recognition group. The probe, LAN-CA, was synthesized by esterifying CA with a near-infrared fluorophore, LAN-OH. This novel fluorescent probe leverages the unique affinity of CA for hepatocytes, ensuring that LAN-CA remains and accumulates specifically within the hepatoenteric circulation. In vitro experiments showed that the probe exhibits superior optical performance compared to traditional benzoate-based probe (LAN-PH), with a detection limit of 0.015 μg/mL. Examination of 56 common biological interferents demonstrated that using CA as a recognition group offers high selectivity. Cell experiments confirmed that LAN-CA is an effective tool for monitoring endogenous CES2 in live cells. Comprehensive evaluations of fluorescence imaging in various mouse models of liver diseases, such as HCC, DILI, and NAFLD, demonstrated that LAN-CA provides exceptional imaging accuracy and therapeutic monitoring capabilities. In conclusion, this probe not only can be a promising tool for accurate liver disease diagnosis, but also can provide valuable insights into treatment efficacy.

Pattern Recognition of Alkaloids by Inhibiting the Catalytic Activity of Dopzymes for Dopamine.

Exploiting the specific recognition probe for all of the biomolecules is difficult in "lock-and-key" biosensors. The cross-reaction or the semispecific probe in pattern recognition mode is an alternative strategy through extracting a multidimensional signal array from recognition elements. Here, we design a pattern recognition sensor array based on the alkaloid-inhibited catalytic activity of dopzymes for the discrimination and determination of six alkaloids. In this sensor array, three different G-rich sequences, i.e., G-triplex (G3), G-quadruplex (GQ1), and the G-quadruplex dimer (2GQ1) possessing various peroxidase activities, conjugated with a dopamine aptamer and the dopzymes (G3-d-apt, GQ1-d-apt, and 2GQ1-d-apt) were obtained with an enhanced catalytic performance for the substrate. Through the interactions between six target alkaloids and G3, GQ1, and 2GQ1 regions, the pattern signal (6 alkaloids × 3 dopzymes × 5 replicates) was obtained from the diverse inhibited effect for the dopzyme activity. In virtue of the statistical method principal component analysis (PCA), the data array was projected into a new dimensional space to acquire the three-dimensional (3D) canonical scores and grouped into their respective clusters. The sensor array exhibited an outstanding discrimination and classification capability for six alkaloids with different concentrations with 100% accuracy. In addition, the nonspecific recognition elements of the sensor array showed high selectivity even though other alkaloids with similar structures to targets existed in the samples. Importantly, the levels of the six targets can be analyzed by the most influential discrimination factor, which represented the vector with the highest variance, evidencing that the sensor array has potential in drug screening and clinical treatment.

Construction and Application of Nanozyme Sensor Arrays.

Compared with traditional "lock-key mode" biosensors, a sensor array consists of a series of sensing elements based on intermolecular interactions (typically hydrogen bonds, van der Waals forces, and electrostatic interactions). At the same time, sensor arrays also have the advantages of fast response, high sensitivity, low energy consumption, low cost, rich output signals, and imageability, which have attracted widespread attention from researchers. Nanozymes are nanomaterials which own enzyme-like properties. Because of the adjustable activity, high stability, and cost effectiveness of nanozymes, they are potential candidates for construction of sensor arrays to output different signals from analytes through the chemoresponse of colorants, which solves the shortcomings of traditional sensors that they cannot support multiple detection and lack universality. Recently, a sensor array based on nanozymes as nonspecific recognition receptors has attracted much more attention from researchers and has been applied to precise recognition of proteins, bacteria, and heavy metals. In this perspective, attention is given to nanozymes and the regulation of their enzyme-like activity. Particularly, the building principles and methods for sensor arrays based on nanozymes are analyzed, and the applications are summarized. Finally, the approaches to overcome the challenges and perspectives are also presented and analyzed for facilitating further research and development of nanozyme sensor arrays. This perspective should be helpful for gaining insight into research ideas within the field of nanozyme sensor arrays.

Molecular Imprinting-Based SERS Detection Strategy for the Large-Size Protein Quantitation and Curbing Non-Specific Recognition.

Molecular imprinting-based surface-enhanced Raman scattering (MI-SERS) sensors have shown remarkable potential from an academic standpoint. However, their practical applications, especially in the detection of large-size protein (≥10 nm), face challenges due to the lack of versatile sensing strategies and nonspecific fouling of matrix species. Herein, we propose a Raman reporter inspector mechanism (RRIM) implemented on a protein-imprinted polydopamine (PDA) layer coated on the SERS active substrate. In the RRIM, after large-size protein recognition, the permeability of the PDA imprinted cavities undergoes changes that are scrutinized by Raman reporter molecules. Target proteins can specifically bind and fully occupy the imprinted cavities, whereas matrix species cannot. Then, Raman reporter molecules with suitable size are introduced to serve as both inspectors of the recognition status and inducers of the SERS signal, which can only penetrate through the vacant and nonspecifically filled cavities. Consequently, changes in the SERS signal exclusively originate from the specific binding of target proteins, while the nonspecific recognition of matrix species is curbed. The RRIM enables reproducible quantitation of the large-size cyanobacteria-specific protein model (≥10 nm), phycocyanin, at the level down to 2.6 × 10-3 μg L-1. Finally, the practical applicability of the RRIM is confirmed by accurately analyzing crude urban waterway samples over 21 min without any pretreatment.

The MRE11–ATM–SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11–ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Targeting Pyroptosis through Lipopolysaccharide-Triggered Noncanonical Pathway for Safe and Efficient Cancer Immunotherapy.

Inducing pyroptosis in cancer cells holds great potential in cancer immunotherapy. Lipopolysaccharide (LPS)-sensing noncanonical pathways are an important mechanism of pyroptosis to eliminate damaged cells, which has not yet been explored for cancer immunotherapy. Here, we utilize bacterial outer membrane vesicles (OMVs) as a natural LPS carrier to trigger a noncanonical pyroptosis pathway for immunotherapy. To address the concern of systemic toxicity, molecule engineered OMVs were designed by equipping DNA aptamers on the OMVs (Apt-OMVs). In addition to improving capacity to target tumors, Apt-OMVs also took advantage of the spherical nucleic acid structure to shield OMVs against nonspecific immune recognition and evade immunogenicity. The selective pyroptosis enhanced tumor immunogenicity, not only promoting the infiltration of effector T cells but also reducing the amount of immunosuppressive regulatory T cells, which remarkably suppressed tumor growth. This work reports the first pyroptosis inducer by the noncanonical pathway, offering inspiration for safe and efficient pyroptosis-mediated immunotherapy.

Non-specific recognition of histone modifications by H3K9bhb antibody

Ketone bodies are short-chain fatty acids produced in the liver during periods of limited glucose availability that provide an alternative energy source for the brain, heart, and skeletal muscle. Beyond this metabolic role, β-hydroxybutyrate (BHB), is gaining recognition as a signaling molecule. Lysine β-hydroxybutyrylation (Kbhb) is a newly discovered post-translational modification in which BHB is covalently attached to lysine ε-amino groups. This protein adduct is metabolically sensitive, dependent on BHB concentration, and found on proteins in multiple intracellular compartments. Therefore, Kbhb is hypothesized to be an important component of ketone body-regulated physiology. Kbhb on histones is proposed to be an epigenetic regulator, which links metabolic alterations to gene expression. However, we found that the widely used antibody against β-hydroxybutyrylated lysine 9 on histone H3 (H3K9bhb) also recognizes other modification(s) that likely include acetylation. Therefore, caution must be used when interpreting gene regulation data acquired with the H3K9bhb antibody.

Upregulation of multiple toll-like receptors in ferret brain after blast exposure: Potential targets for treatment

Although blast-induced traumatic brain injury (bTBI) has been designated as the signature injury of recent combat operations, its precise pathological mechanism(s) has not been identified thus far. Prior preclinical studies on bTBI demonstrated acute neuroinflammatory cascades which are known to be contributing to neurodegeneration. Danger-associated chemical patterns are released from the injured cells, which activate non-specific pattern recognition receptors, such as toll-like receptors (TLRs) leading to increased expression of inflammatory genes and release of cytokines. Upregulation of specific TLRs in the brain has been described as a mechanism of injury in diverse brain injury models unrelated to blast exposure. However, the expression profile of various TLRs in bTBI has not been investigated thus far. Hence, we have evaluated the expression of transcripts for TLR1-TLR10 in the brain of a gyrencephalic animal model of bTBI. We exposed ferrets to tightly coupled repeated blasts and determined the differential expression of TLRs (TLR1-10) by quantitative RT-PCR in multiple brain regions at 4 hr, 24 hr, 7 days and 28 days post-blast injury. The results obtained indicate that multiple TLRs are upregulated in the brain at 4 hr, 24 hr, 7 days and 28 days post-blast. Specifically, upregulation of TLR2, TLR4 and TLR9 was noted in different brain regions, suggesting that multiple TLRs might play a role in the pathophysiology of bTBI and that drugs that can inhibit multiple TLRs might have enhanced efficacy to attenuate brain damage and thereby improve bTBI outcome. Taken together, these results suggest that several TLRs are upregulated in the brain after bTBI and participate in the inflammatory response and thereby provide new insights into the disease pathogenesis. Therefore, inhibition of multiple TLRs, including TLR2, 4 and 9, simultaneously might be a potential therapeutic strategy for the treatment of bTBI.

Peptide functionalized nanocarriers for targeted drug delivery in cancer therapy.

The intrinsic limitations of conventional cancer therapies have prompted the development of nanocarriers for more effective and safer cancer treatment. Amongst these, nanocarriers functionalized with tumor-targeting moieties have been utilized to enhance delivery of cancer therapeutics. However, recent reports have revealed that only a modest increase in tumor targeting is typically achieved with this approach. Poor tumor accumulation is largely due to formation of a serum protein corona on the surface of the nanocarriers in circulation, which hinders targeting and leads to nonspecific tissue distribution, immune recognition and rapid blood clearance. Here, we present three highly efficient tumor-targeting nanocarriers that overcome the aforementioned issues. These nanocarriers are functionalized with the acidity-triggered rational membrane (ATRAM) peptide that facilitates internalization specifically into cancer cells within the acidic tumor microenvironment. The first system is a hybrid nanocarrier that consists of a drug-loaded polylactic-co-glycolic acid (PLGA) core covalently ‘wrapped’ with a crosslinked bovine serum albumin (BSA) shell. Second are upconversion mesoporous silica nanospheres (UMSNs), with combined photodynamic therapy, photothermal therapy and imaging capabilities, that consist of a lanthanide-doped core ‘wrapped’ with lipid-polyethylene glycol (DSPE-PEG-maleimide). The final system consists of drug-loaded mesoporous silica nanoparticles (MSNs) coated with a lipid bilayer composed of DPPC/Chol/DSPE-PEG-mal. The lipid coat is then functionalized with programmed death receptor ligand-1 antibody (anti-PD-L1 antibody, atezolizumab) to disrupt the programmed death 1 (PD-1) interaction that inhibits the anticancer activity of tumor-infiltrating immune cells. In all three systems, the BSA or lipid coats serve to minimize interactions with serum proteins, which allows the nanocarriers to evade immune recognition and extends the in vivo circulation half-life. All three systems exhibit remarkable tumor-targeting efficiency leading to highly potent anticancer activity, while exhibiting negligible toxicity to healthy tissue. Thus, all three systems represent highly promising nanoplatforms for the targeted delivery of cancer therapeutics.

A pH-Responsive Covalent Nanoscale Device Enhancing Temporal and Force Stability for Specific Tumor Imaging.

Approaches to DNA probe-mediated precision medicine have been extensively explored for the diagnosis and treatment of diverse types of cancer. Despite this, simple nanoscale devices with the required recognition specificity and sensitivity for clinical application have remained elusive until now. Here, we report a pH-driven covalent nanoscale device that integrates pH-responsive, switchable structure and proximity-driven covalent cross-linking. A tumor acidic, pH-driven mechanism eliminates "on-target, off-tumor" nonspecific recognition. By manipulating covalent binding to target molecule on the cell surface, this nanodevice avoids binding-then-shedding to improve the sensitivity of tumor recognition. We envision that this pH-driven covalent nanoscale device will inspire more clinical applications toward specific, long-term tumor imaging in the cancer microenvironment.

Chiral molecular imprinting-based SERS detection strategy for absolute enantiomeric discrimination

Chiral discrimination is critical in environmental and life sciences. However, an ideal chiral discrimination strategy has not yet been developed because of the inevitable nonspecific binding entity of wrong enantiomers or insufficient intrinsic optical activities of chiral molecules. Here, we propose an “inspector” recognition mechanism (IRM), which is implemented on a chiral imprinted polydopamine (PDA) layer coated on surface-enhanced Raman scattering (SERS) tag layer. The IRM works based on the permeability change of the imprinted PDA after the chiral recognition and scrutiny of the permeability by an inspector molecule. Good enantiomer can specifically recognize and fully fill the chiral imprinted cavities, whereas the wrong cannot. Then a linear shape aminothiol molecule, as an inspector of the recognition status is introduced, which can only percolate through the vacant and nonspecifically occupied cavities, inducing the SERS signal to decrease. Accordingly, chirality information exclusively stems from good enantiomer specific binding, while nonspecific recognition of wrong enantiomer is curbed. The IRM benefits from sensitivity and versatility, enabling absolute discrimination of a wide variety of chiral molecules regardless of size, functional groups, polarities, optical activities, Raman scattering, and the number of chiral centers.

The role of PD-1/PD-L1 inhibitors in the treatment for non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) is a frequent cancer that affects people around the world. Checkpoint inhibitors are the most intensively studied treatment methods at present. Activation of PD-1/PD-L1 is accountable for suppressing the activation, proliferation, and cytotoxic secretion of T cells. By giving PD-1 /PD-L1 inhibitors, an individual's immune system can be boosted to some extent. Nivolumab, pembrolizumab, atezolizumab, and durvalumab have shown to be effective for NSCLC patients in clinical trials. The effectiveness of PD-1/PD-L1 inhibitors combining with SBRT, chemotherapy, or other checkpoint inhibitors to treat NSCLC has also been demonstrated. However, PD-1/PD-L1 inhibitors also have drawbacks, such as non-specific recognition misses, and immune-related adverse events. This article mainly discusses the role of PD-1/PD-L1 inhibitors in the treatment for NSCLC.

Human motion intention recognition method with visual, audio, and surface electromyography modalities for a mechanical hand in different environments

Mechanical hand is widely used in industrial and rehabilitation fields. Good interaction will enable it to execute human ideas quickly and complete related tasks with humans well. Different environments will affect its performance. Therefore, the modality diversity and environmental adaptability of human–machine interaction are important.In this study, a visual modality was developed based on Mediapipe framework and Tensorflow lite. An audio modality was developed based on Chinese finger-guessing game terms and non-specific voice recognition technology. A surface electromyography (sEMG) modality was developed based on machine learning, and a touch interface was developed based on serial touch screen. Furthermore, the user can choose different modalities according to the environment through the touch screen.The experimental results show that the average accuracies of visual modality, audio modality, and sEMG modality are 98.2463%±1.5057%, 97.3132%±0.692%, and 96.3454%±2.0108%, respectively. On a computer equipped with Intel (R) Core (TM) i5-1137G7 CPU, the execution time of visual modality, audio modality, and sEMG modality for a single action in Python 3.6 are 0.03 s, 0.81 s, and 0.19 s, respectively.Compared with several existing methods, the proposed method has rich modalities, better accuracy, well real-time performance of motion recognition, and real-time modality switching function.

ROS responsive nanoparticles loaded with lipid-specific AIEgen for atherosclerosis-targeted diagnosis and bifunctional therapy

Atherosclerosis, which is triggered by endothelial damage, progressive local inflammation and excessive lipid accumulation, is one of the most common cardiovascular diseases in recent years. Drug delivery systems have shown great potential for the accurate diagnosis and effective treatment of early atherosclerosis, but are accompanied by disadvantages such as poor stability, lack of active targeting and non-specific recognition capabilities, which still need to be further developed. In our work, a multifunctional nanoparticle (LFP/PCDPD) with reactive oxygen species (ROS) responsive drug release, lipid removal, and lipid-specific AIE fluorescence imaging was constructed. Cyclodextrin structure with lipid removal function and PMEMA blocks with ROS-response-mediated hydrophobic to hydrophilic conversion were simultaneously introduced into the structure of LFP/PCDPD to load the anti-inflammatory drug prednisolone (Pred) and lipid-specific AIEgen (LFP). The active targeting function of LFP/PCDPD was conferred by the high affinity of dextran to the vascular adhesion molecule-1 (VCAM-1) and CD44 receptor on the surface of broken endothelial cells. After intravenous injection into ApoE−/− mice, LFP/PCDPD actively enriched in the microenvironment of local ROS overexpression and rich lipids in atherosclerosis. Pred and LFP were released while lipids were removed, thus enabling proactive targeting of atherosclerosis and efficient “two-pronged” treatment.

Pathophysiology of RAGE in inflammatory diseases.

The receptor for advanced glycation end products (RAGE) is a non-specific multi-ligand pattern recognition receptor capable of binding to a range of structurally diverse ligands, expressed on a variety of cell types, and performing different functions. The ligand-RAGE axis can trigger a range of signaling events that are associated with diabetes and its complications, neurological disorders, cancer, inflammation and other diseases. Since RAGE is involved in the pathophysiological processes of many diseases, targeting RAGE may be an effective strategy to block RAGE signaling.

Synergetic regulation of kupffer cells, extracellular matrix and hepatic stellate cells with versatile CXCR4-inhibiting nanocomplex for magnified therapy in liver fibrosis

Hepatic stellate cell (HSC)-targeted delivery is an attractive strategy for liver fibrosis therapy, but the efficacy is hampered by poor delivery of nanomaterials and complicated microenvironments of the fibrotic liver. Here, we report a versatile CXCR4-inhibiting nanocomplex composed of polymeric CXCR4 antagonism (PAMD, PA), CLD (clodronate) and siPAI-1 (siRNA of plasminogen activator inhibitor-1) that surmounts multiple barriers to improve the outcome by co-regulating Kupffer cells (KCs), extracellular matrix (ECM) and HSCs. Upon encountering biological barriers, the nanocomplex exerted penetrating and targeting functions, efficiently overcoming KCs capture, ECM trapping and nonspecific recognition of HSCs, finally contributing to the enhanced HSCs uptake. Moreover, an enlarged antifibrotic activity is realized through synergetic regulation of KCs apoptosis, ECM degradation and HSCs inactivation. Overall, such a versatile nanocomplex provides a framework for designing HSC-targeted delivery system and has valuable potential as a novel antifibrotic strategy.

CAR-T Regulatory (CAR-Treg) Cells: Engineering and Applications.

Regulatory T cells are critical for maintaining immune tolerance. Recent studies have confirmed their therapeutic suppressive potential to modulate immune responses in organ transplant and autoimmune diseases. However, the unknown and nonspecific antigen recognition of polyclonal Tregs has impaired their therapeutic potency in initial clinical findings. To address this limitation, antigen specificity can be conferred to Tregs by engineering the expression of transgenic T-cell receptor (TCR) or chimeric antigen receptor (CAR). In contrast to TCR Tregs, CAR Tregs are major histocompatibility complex (MHC) independent and less dependent on interleukin-2 (IL-2). Furthermore, CAR Tregs maintain Treg phenotype and function, home to the target tissue and show enhanced suppressive efficacy compared to polyclonal Tregs. Additional development of engineered CAR Tregs is needed to increase Tregs’ suppressive function and stability, prevent CAR Treg exhaustion, and assess their safety profile. Further understanding of Tregs therapeutic potential will be necessary before moving to broader clinical applications. Here, we summarize recent studies utilizing CAR Tregs in modulating immune responses in autoimmune diseases, transplantation, and gene therapy and future clinical applications.

Differential biomolecular recognition by synthetic vs. biologically-derived components in the stone-forming process using 3D microfluidics.

Calcium phosphate (CaP) biomineralization is the hallmark of extra-skeletal tissue calcification and renal calcium stones. Although such a multistep process starts with CaP crystal formation, the mechanism is still poorly understood due to the complexity of the in vivo system and the lack of a suitable approach to simulate a truly in vivo-like environment. Although endogenous proteins and lipids are engaged with CaP crystals in such a biological process of stone formation, most in vitro studies use synthetic materials that can display differential bioreactivity and molecular recognition by the cellular component. Here, we used our in vitro microfluidic (MF) tubular structure, which is the first completely cylindrical platform, with renal tubular cellular microenvironments closest to the functional human kidney tubule, to understand the precise role of biological components in this process. We systematically evaluated the contribution of synthetic and biological components in the stone-forming process in the presence of dynamic microenvironmental cues that originated due to cellular pathophysiology, which are critical for the nucleation, aggregation, and growth of CaP crystals. Our results show that crystal aggregation and growth were enhanced by immunoglobulin G (IgG), which was further inhibited by etidronic acid due to the chelation of extracellular Ca2+. Interestingly, biogenic CaP crystals from mice urine, when applied with cell debris and non-specific protein (bovine serum albumin), exhibited a more discrete crystal growth pattern, compared to exposure to synthetic CaP crystals under similar conditions. Furthermore, proteins found on those calcium crystals from mice urine produced discriminatory effects on crystal-protein attachment. Specifically, such biogenic crystals exhibited enhanced affinity to the proteins inherent to those crystals. More importantly, a physiological comparison of crystal induction in renal tubular cells revealed that biogenic crystals are less effective at producing a sustained rise in cytosolic Ca2+ compared to synthetic crystals, suggesting a milder detrimental effect to downstream signaling. Finally, synthetic crystal-internalized cells induced more oxidative stress, inflammation, and cellular damage compared to the biogenic crystal-internalized cells. Together, these results suggest that the intrinsic nature of biogenically derived components are appropriate to generate the molecular recognition needed for spatiotemporal effects and are critical towards understanding the process of kidney stone formation.