RESUMO
Ferroptosis and apoptosis are two types of regulated cell death that are closely associated with the pathophysiological processes of many diseases. The significance of ferroptosis-apoptosis crosstalk in cell fate determination has been reported, but the underlying molecular mechanisms are poorly understood. Herein mitochondria-mediated molecular crosstalk is explored. Based on a comprehensive spectroscopic investigation and mass spectrometry, cytochrome c-involved Fenton-like reactions and lipid peroxidation are revealed. More importantly, cytochrome c is found to induce ROS-independent and cardiolipin-specific lipid peroxidation depending on its redox state. In situ Raman spectroscopy unveiled that erastin can interrupt membrane permeability, specifically through cardiolipin, facilitating cytochrome c release from the mitochondria. Details of the erastin-cardiolipin interaction are determined using molecular dynamics simulations. This study provides novel insights into how molecular crosstalk occurs around mitochondrial membranes to trigger ferroptosis and apoptosis, with significant implications for the rational design of mitochondria-targeted cell death reducers in cancer therapy.
Assuntos
Ferroptose , Análise Espectral Raman , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Mitocôndrias/metabolismo , Peroxidação de LipídeosRESUMO
The selective interaction of cytochrome c (Cyt c) with cardiolipin (CL) is involved in mitochondrial membrane permeabilization, an essential step for the release of apoptosis activators. The structural basis and modulatory mechanism are, however, poorly understood. Here, we report that Cyt c can induce CL peroxidation independent of reactive oxygen species, which is controlled by its redox states. The structural basis of the Cyt c-CL binding was unveiled by comprehensive spectroscopic investigation and mass spectrometry. The Cyt c-induced permeabilization and its effect on membrane collapse, pore formation, and budding are observed by confocal microscopy. Moreover, cytochrome c oxidase dysfunction is found to be associated with the initiation of Cyt c redox-controlled membrane permeabilization. These results verify the significance of a redox-dependent modulation mechanism at the early stage of apoptosis, which can be exploited for the design of cytochrome c oxidase-targeted apoptotic inducers in cancer therapy.
Assuntos
Citocromos c , Análise Espectral Raman , Citocromos c/química , Citocromos c/metabolismo , Citocromos c/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Oxirredução , Cardiolipinas/química , Cardiolipinas/metabolismo , Cardiolipinas/farmacologia , Membranas Mitocondriais/metabolismo , ApoptoseRESUMO
In situ analysis of membrane protein-ligand interactions under physiological conditions is of significance for both fundamental and applied science, but it is still a big challenge due to the limits in sensitivity and selectivity. Here, we demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for the investigation of membrane protein-protein interactions. Lipid biolayers are successfully coated on silver nanoparticles through electrostatic interactions, and a highly sensitive and biomimetic membrane platform is obtained in vitro. Self-assembly and immobilization of the reduced cytochrome b5 on the coated membrane are achieved and protein native biological functions are preserved. Owing to resonance effect, the Raman fingerprint of the immobilized cytochrome b5 redox center is selectively enhanced, allowing for in situ and real-time monitoring of the electron transfer process between cytochrome b5 and their partners, cytochrome c and myoglobin. This study provides a sensitive analytical approach for membrane proteins and paves the way for in situ exploration of their structural basis and functions.
Assuntos
Nanopartículas Metálicas , Análise Espectral Raman , Proteínas de Membrana , Elétrons , Citocromos b , Prata/químicaRESUMO
The crosstalk between mitochondria and endoplasmic reticula plays a crucial role in apoptotic pathways in which reactive oxygen species (ROS) produced by microsomal monooxygenase (MMO) are believed to accelerate cytochrome c release. Herein, we successfully demonstrate the potential of surface-enhanced resonance Raman spectroscopy (SERRS) for monitoring MMO-derived ROS formation and ROS-mediated cytochrome c release. Silver nanoparticles coated with nickel shells are used as both Raman signal enhancers and electron donors for cytochrome c. SERRS of cytochrome c is found to be sensitive to ROS, allowing for in situ probing of ROS formation with a cell death inducer. Label-free evaluation of ROS-induced apoptosis is achieved by SERRS-based monitoring of cytochrome c release in living cells. This study verifies the capability of SERRS for label-free, in situ, and real-time monitoring of the mitochondria-endoplasmic reticulum crosstalk in apoptosis and provides a novel strategy for the rational design and screening of ROS-inducing drugs for cancer treatment.
Assuntos
Nanopartículas Metálicas , Análise Espectral Raman , Citocromos c , Espécies Reativas de Oxigênio , Prata/farmacologia , Retículo Endoplasmático , Mitocôndrias , ApoptoseRESUMO
Sensitive and multiple detection of the biomarkers of type 1 diabetes mellitus (T1DM) is vital to the early diagnosis and clinical treatment of T1DM. Herein, we developed a SERS-based biosensor using polyvinylidene fluoride (PVDF) membranes as a flexible support for the detection of glutamic acid decarboxylase antibodies (GADA) and insulin autoantibodies (IAA). Two kinds of silver-gold core-shell nanotags embedded with Raman probes and attached with GADA or IAA antibodies were synthesized to capture the targets, enabling highly sensitive and highly selective detection of GADA and IAA. The embedded Raman probes sandwiched between silver and gold layers guaranteed spectral stability and reliability. Moreover, the utilization of two Raman probes enables simultaneous and multiplexing detection of both GADA and IAA, improving the detection accuracy for T1DM. The proposed SERS-based method has been proven feasible for clinical sample detection, demonstrating its great potential in sensitive, reliable, and rapid diagnosis of T1DM.
Assuntos
Técnicas Biossensoriais , Diabetes Mellitus Tipo 1 , Nanopartículas Metálicas , Humanos , Diabetes Mellitus Tipo 1/diagnóstico , Prata , Reprodutibilidade dos Testes , Biomarcadores , Anticorpos , Ouro , Análise Espectral Raman/métodosRESUMO
In multienzymes cascade reaction, the inter-enzyme spacing is supposed to be a factor affecting the cascade activity. Here, a simple and efficient Y-shaped DNA scaffold is assembled using two partially complementary DNA single strands on magnetic microspheres, which is used to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP). As a result, on poly(vinyl acetate) magnetic microspheres (PVAC), GOD/HRP-DNA@PVAC multienzyme system is obtained, which can locate GOD and HRP accurately and control the inter-enzyme distance precisely. The distance between GOD and HRP is regulated by changing the length of DNA strand. It showed that the cascade activity is significantly distance-dependent. Moreover, the inter-enzyme spacing is not the closer the better, and too short distance would generate steric hindrance between enzymes. The cascade activity reached the maximum value of 967 U mg-1 at 13.6 nm, which is 3.5 times higher than that of free enzymes. This is ascribed to the formation of substrate channeling.
Assuntos
Enzimas Imobilizadas , Glucose Oxidase , Peroxidase do Rábano Silvestre , Microesferas , DNARESUMO
Graphene oxide derivatives (GODs) have superb physical/chemical properties with promise for applications in biomedicine. Shape, size, and chemistry of the GODs are identified as the key parameters that impact any biological system. In this work, the GODs with a wide range of shapes (sheets, helical/longitudinal ribbons, caps, dots), sizes (10 nm to 20 µm), and chemistry (partially to fully oxidized) are synthesized, and their cytotoxicity in normal cells (NIH3T3) and colon cancer cells (HCT116) are evaluated. The mechanisms by which the GODs induce cytotoxicity are comprehensively investigated, and the toxic effects of the GODs on the NIH3T3 and the HCT116 cells are compared. While the GODs show no toxicity under the size of 50 nm, they impose moderate toxic effects at the sizes of 100 nm to 20 µm (max viability >57%). For the GODs with the similar size (100-200 nm), the helical ribbon-like structure is found to be much less toxic than the longitudinal ribbon structure (max viability 83% vs 18%) and the tubular structure (0% viability for the oxidized carbon nanotubes). It is also evident that the level of oxidation of the GOD is inversely related to the toxicity. Although the extent of GOD-induced cytotoxicity (reduction of cell viability) to the two cell lines is similar, their toxicity mechanisms are interestingly found to be substantially different. In the HCT116 cancer cells, cell membrane leakage leads to DNA damage followed by cell death, whereas in the NIH3T3 normal cells, increases in oxidative stress and physical interference between the GODs and the cells are identified as the main toxicity sources.
Assuntos
Grafite/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Grafite/química , Células HCT116 , Humanos , Camundongos , Células NIH 3T3 , Tamanho da PartículaRESUMO
Interfaces play an important role in enhancing the energy conversion performance of dye-sensitized solar cells (DSCs). The interface effects have been studied by many techniques, but most of the studies only focused on one part of a DSC, rather than on a complete solar cell. Hence, monitoring the interface evolution of a DSC is still very challenging. Here, inâ situ/operando resonance Raman (RR) spectroscopic analyses were carried out to monitor the dynamics of the photovoltaic conversion processes in a DSC. We observed the creation of new species (i.e., polyiodide and iodine aggregates) in the photosensitization process. We also obtained molecular-scale dynamic evidence that the bands from the C=C and C=N bonds of 2,2'-bipyridyl (bpy), the S=C=N bonds of the NCS ligand, and photochemical products undergo reasonably strong intensity and frequency changes, which clearly demonstrates that they are involved in charge separation. Furthermore, RR spectroscopy can also be used to quickly evaluate the performance of DSCs.
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Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in protein identification and quantification. However, the poor spectral reproducibility, originating from random protein immobilization on SERS substrates, still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels. Here, in our study, spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement. We have accordingly prepared iminodiacetic acid (IDA)-functionalized silver substrates, which are used for capturing His-tagged proteins via nickel-imidazole coordination. The controlled immobilization enables excellent SERS spectral reproducibility as evidenced by 6 polypeptides. Furthermore, the interactions between two model proteins, Erv1C (C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1) and AFP (alpha-fetoprotein), and their ligands Cyt c (cytochrome c) and ATRA (all-trans-retinoic acid) are examined, respectively. The results indicate that the IDA-functionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS. As a proof-of-concept study, the proposed functionalized SERS-active substrates combined with immobilized metal-affinity chromatography will be useful for mechanism studies on protein-ligand interactions, which is crucially important for understanding the structural basis of protein functional versatility and will contribute to the fields of drug design and biotechnology.
Assuntos
Proteínas/metabolismo , Análise Espectral Raman/métodos , Animais , Citocromos c/metabolismo , Humanos , Iminoácidos/química , Proteínas Imobilizadas/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Prata/química , Propriedades de Superfície , alfa-Fetoproteínas/metabolismoRESUMO
Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in label-free DNA detection. Owing to the limitation in chain length, it is however still challenging for SERS as a routine method to explore the intrinsic structural information on unmodified DNA. Here, we develop a universal SERS-based approach toward quantification of A/G in single-stranded DNAs (12 up to 28 bases) by introducing a novel interfacial agent, dichloromethane. DNA hybridization is successfully probed as evidenced by the typical SERS bands attributed to hydrogen bonds in a hairpin structure. More importantly, enlarged space of "hot spots" in SERS enables discrimination of single-base mutation in double-stranded DNA with 100 bases, which as a proof-of-concept study will pave a new avenue for highly sensitive DNA detection in clinical applications.
Assuntos
DNA/genética , Mutação Puntual , Análise Espectral Raman/métodos , Sequência de Bases , DNA/análise , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , Indicadores e Reagentes , Cloreto de Metileno , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodosRESUMO
Intrinsic properties of nickel have enabled its wide applications as an effective catalyst. In this study, nickel nanowires (Ni NWs) as electron donors for oxidized cytochrome c (Cyt c) are investigated, which are NW diameter, temperature, and pH value-dependent. The reductive and magnetic properties facilitate the Ni NWs to rapidly and conveniently reduce Cyt c in complicated biological samples. Moreover, we find that the Ni NWs combined with resonance Raman spectroscopy have specificity toward Cyt c detection in real biological samples, which is successfully used to distinguish the redox state of the released Cyt c from isolated mitochondria in apoptotic Hela cells. Moreover, rapid label-free Cyt c quantification can be achieved by surface-enhanced Raman spectroscopy with a limit of detection of 1 nM and long concentration linear range (1 nM-1 µM). The proposed Ni NWs-based reduction approach will significantly simplify the traditional biological methods and has great potential in the application of Cyt c-related apoptotic studies.
Assuntos
Apoptose/fisiologia , Citocromos c/análise , Nanofios/química , Níquel/química , Citocromos c/química , Citocromos c/metabolismo , Células HeLa , Humanos , Limite de Detecção , Mitocôndrias/metabolismo , Oxirredução , Análise Espectral Raman/métodosRESUMO
Frequency-shift based surface-enhanced Raman spectroscopy (SERS) has exhibited great potential applications in bioanalytical chemistry and biomedicine in recent years. The basis and the crucial factors determining frequency shifts are, however, still unclear. Herein, we have systematically investigated how solvents, antigens, and antibodies affect the band shifts in SERS-based immunoassays. By applying the charge transfer theory together with the Stark effect and time-dependent density functional theory (TDDFT) calculation, mechanistic insights into the frequency shifts in immunoreactions is proposed and discussed in detail. Accordingly, the experimental condition is further optimized and is successfully applied for the first time to detect carbonylated proteins, promising diagnostic biomarkers for human diseases. This study provides theoretical guidance for designing SERS frequency shift-based immunoassays and paves a new avenue for further applications of the strategy in clinical diagnosis.
Assuntos
Imunoensaio/métodos , Carbonilação Proteica , Análise Espectral Raman/métodos , Biomarcadores/análise , Teoria da Densidade Funcional , Humanos , Modelos TeóricosRESUMO
The interaction of cytochromeâ c (Cytâ c) with cardiolipin (CL) is believed to play an important role in the initial events of apoptosis. Herein, we investigate the structural changes of CL-bound Fe2+ Cyt c and the correlation with Cytâ c release through surface-enhanced Raman spectroscopy (SERS) on nickel substrates. The SERS results together with molecular dynamics simulation reveal that Fe2+ Cytâ c undergoes autoxidation and a relatively larger conformational alteration after binding with CL, inducing higher peroxidase activity of Cytâ c and higher permeability of the CL membrane compared with those induced by the Fe3+ Cytâ c. The proapoptotic activity and SERS effect of the Ni nanostructures allow the inâ situ study of the redox-state-dependent Cyt c release from isolated mitochondria, which reveals for the first time that the ferrous state of Cytâ c most likely plays a more important role in triggering apoptosis.
Assuntos
Apoptose , Citocromos c/metabolismo , Níquel/metabolismo , Sítios de Ligação , Cardiolipinas/química , Cardiolipinas/metabolismo , Citocromos c/química , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Nanoestruturas/química , Nanoestruturas/toxicidade , Níquel/química , Oxirredução , Peroxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise Espectral RamanRESUMO
Protein biomarkers are very important indicators of diseases and have great potential in cancer early diagnosis. The majority of detection methods for protein biomarkers currently rely on specific capture antibodies or aptamers with chemiluminescent and fluorescent labels. Here, an antibody-free strategy for discrimination of versatile proteins is proposed based on surface-enhanced Raman spectroscopy. The SERS spectral variation of a linker molecule, perylenetetra carboxylic acid (PTCA), is found to directly correlate with the protein types, according to which protein biomarkers even homologous proteins with very similar molecular structures can be discriminated with the aid of hierarchical cluster analysis. Furthermore, the feasibility of the proposed approach has been proved in early liver cancer diagnosis with clinical samples. All the results indicate that PTCA as a universal SERS probe has great potential in rapid, accurate, and direct protein biomarker discrimination in cancer diagnosis.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Hepáticas/diagnóstico , Proteínas de Neoplasias/sangue , Ácidos Carboxílicos/química , Humanos , Neoplasias Hepáticas/sangue , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
Charge transfer (CT) at the interfaces between titanium dioxide (TiO2) and gold (Au) is investigated by surface-enhanced Raman scattering (SERS) spectroscopy probed by a sandwiched molecule 4-mercaptobenzoic acid (4-MBA). For the first time, the contribution of surface plasmon resonance (SPR) to CT is studied by tuning the surface plasmon absorption of Au nanorods (NRs) from 530 nm to 793 nm. Moreover, the degrees of CT in the TiO2-MBA-Au assemblies are calculated and the maximum degree of CT is obtained when the excitation laser wavelength is resonant with the SPR absorption of the assemblies. Accordingly, we propose a CT pathway in these semiconductor-molecule-metal assemblies, and the mechanism by which SPR contributes to the CT at the interfaces is discussed. This study has established a simple and effective way of studying the influence of SPR on interfacial CT by using SERS, which is beneficial for further investigations on interfacial charge transfers. Our findings will have significant importance for the improvement of photoelectric devices and photocatalytic efficiency.
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α-Fetoprotein (AFP) is an important tumor biomarker. In particular, the overexpression of AFP-L3 is associated with hepatocellular carcinoma (HCC). Accordingly, several hospitals have begun to employ the ratio of AFP-L3 to the total AFP level (AFP-L3%) as new diagnostic evidence for HCC owing to its high diagnostic accuracy. However, current methods of detection for AFP and AFP-L3 are time-consuming, require multiple samples, and lack in sensitivity and specificity. Herein, we present a novel concept for the early diagnosis of HCC based on the combination of Raman frequency shift and intensity change, and developed surface-enhanced Raman scattering (SERS)-based immunochips via AFP-L3%. In the first step of the study, the frequency shift of 4-mercaptobenzoic acid (MBA) was applied for the quantitative determination of total AFP based on the AFP and anti-AFP interaction on MBA-modified silver chips. 5,5-Dithiobis(succinimidyl-2-nitrobenzoate) (DSNB)-modified immunogold was then incorporated with AFP-L3 antibodies for sandwich immunoreaction on the chips. As a result, we found that a typical Raman band intensity of DSNB presented an exponential linear relationship with the concentration of AFP-L3. Thus, the AFP-L3% can be calculated according to the concentrations of AFP-L3 and total AFP. The most important advantage of the proposed method is the combination of AFP-L3% and frequency shifts of SERS, which exhibits excellent reproducibility and high accuracy, and significantly simplifies the conventional detection procedure of AFP-L3%. Application of the proposed method with the serum of patients with HCC demonstrated its great potential in early liver cancer diagnosis.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Análise Espectral Raman/métodos , alfa-Fetoproteínas/análise , Anticorpos/imunologia , Benzoatos/química , Detecção Precoce de Câncer , Ouro/química , Humanos , Imunoensaio , Masculino , Nanopartículas Metálicas/química , Pessoa de Meia-Idade , Compostos de Sulfidrila/química , alfa-Fetoproteínas/imunologiaRESUMO
Surface-enhanced Raman spectroscopy (SERS) represents a powerful approach for studying the structure and reaction of proteins in fundamental and applied sciences. The surface properties of SERS-active materials determine important parameters such as Raman enhancement ability, biocompatibility, and electronic communication between supports and proteins. Here, electron transfer (ET) of Cytâ c on noble metals and transition metals is investigated by SERS spectroscopy. The results here indicate that the ET occurs from the reduced state of Cytâ c to silver substrate, depending on the laser wavelengths. Nickel and cobalt can directly transfer electrons to the oxidized state of Cytâ c, which enables a reductive activity of these transition metal nanoparticles (NPs). This study demonstrates the role of transition metals as electron donors for Cytâ c and has proved that the charge transfer theory for SERS is applicable for explanation of the ET between Cytâ c and Ag NPs.
Assuntos
Citocromos c/química , Teste de Materiais/métodos , Análise Espectral Raman/métodos , Transporte de Elétrons , Lasers , Nanopartículas Metálicas/química , Oxirredução , Conformação Proteica , Propriedades de Superfície , Elementos de Transição/químicaRESUMO
We proposed a novel readout method based on a peculiar phenomenon in which the vibrational frequencies of a SERS-active probe (dimethyldithiocarbamic acid sodium salt, DASS) can be affected when there is mercury species. Compared to the SERS intensity-dependent quantitative determination method, SERS frequency-shift-based methods have several advantages: smaller standard deviation, perfect linear relationship, and higher accuracy and sensitivity. In addition, the SERS frequency-shift-based method was not affected by irreproducible aggregation of the SERS substrate and instrumental factors, which greatly improved the application prospect of SERS-based detection. The DASS-modified silver nanoparticles produced a highly sensitive sensor specific to mercury species. Upon the addition of a solution of mercury species to the chip, the mercury species specifically binds to the sulfur atoms, which induces a frequency shift of the band at 1374 cm(-1). The detection limit of the proposed method for Hg(2+) is as low as 10(-8) M. In addition, the proposed method exhibited the same phenomenon for organic mercury. Moreover, these results suggest that the proposed platform possesses the potential for sensitive, selective, and high-throughput on-site mercury pollution monitoring in resource-constrained settings.
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Surface-enhanced resonance Raman scattering (SERRS) has been used to establish a rapid and quantitative assay based on the diazotization coupling reaction for thyrotropin-releasing hormone (TRH). Ultrahigh sensitivity of this approach originates from two factors: changing TRH to an azo compound and the SERRS effect with the addition of silver nanoparticles (AgNPs) at 532 nm excitation wavelength. The lowest detectable concentration of TRH was found to be as low as 1 pg mL(-1), which is 10-fold lower than the lowest normal reference value in human serum reported in previous literature. The quantitative measurements in human serum based on this method were conducted, and the results showed its feasibility for detection in complex biological samples. In comparison with conventional TRH identification and quantification methodologies, radioimmunoassay (RIA) and subsequent various hyphenated techniques, the main advantages of this study are simplicity, rapidness (2 minutes), time effectiveness, no additional steps required to further characterize the immunogenic material, highest sensitivity (57.1 fg), high selectivity, practicality and reliability. Thus, this work puts forward a research tool that may be applied to the determination of TRH in practical assays.
Assuntos
Nanopartículas Metálicas , Análise Espectral Raman , Hormônio Liberador de Tireotropina/sangue , Humanos , Reprodutibilidade dos Testes , PrataRESUMO
This article outlines the recent progress in surface enhanced Raman spectroscopy (SERS) based proteins applications. SERS is a specific Raman spectroscopic technique that provides enhanced Raman signals (several orders of magnitude greater than normal) for numerous Raman-active analyte molecules adsorbed onto rough metal surfaces. SERS is a sensitive, selective, and versatile technique that lends to fast data acquisition in actual time. Therefore, SERS has undergone rapid development because of its technical advantages in instrumentation, data analysis methods and its multiple biological applications. This article highlights several representative areas in proteins where SERS could be employed. Some of the proteins applications of SERS are more maturely developed, whereas others are in their initial stages of development (in laboratories). This article discusses the recent developments in SERS based quantitative analysis: directly with different substrates (e.g. biomolecules on electrodes, colloidal particles, and periodic pattern structure and tip based substrates). Furthermore, SERS based techniques are advantageous for obtaining valuable information on protein-protein, protein-ligand, and protein-drug recognitions via spectral differences among molecular bridges. SERS based techniques show considerable promise for qualitative and/or quantitative analyses of biological systems.