key: cord-0769188-9lwr6k8p
authors: Aitekenov, Sultan; Gaipov, Abduzhappar; Bukasov, Rostislav
title: Review: Detection and quantification of proteins in human urine
date: 2020-10-14
journal: Talanta
DOI: 10.1016/j.talanta.2020.121718
sha: 4a098c0e0db34f3220b3562a9650a83b40c3a8e9
doc_id: 769188
cord_uid: 9lwr6k8p

Extensive medical research showed that patients, with high protein concentration in urine, have various kinds of kidney diseases, referred to as proteinuria. Urinary protein biomarkers are useful for diagnosis of many health conditions – kidney and cardio vascular diseases, cancers, diabetes, infections. This review focuses on the instrumental quantification (electrophoresis, chromatography, immunoassays, mass spectrometry, fluorescence spectroscopy, the infrared spectroscopy, and Raman spectroscopy) of proteins (the most of all albumin) in human urine matrix. Different techniques provide unique information on what constituents of the urine are. Due to complex nature of urine, a separation step by electrophoresis or chromatography are often used for proteomics study of urine. Mass spectrometry is a powerful tool for the discovery and the analysis of biomarkers in urine, however, costs of the analysis are high, especially for quantitative analysis. Immunoassays, which often come with fluorescence detection, are major qualitative and quantitative tools in clinical analysis. While Infrared and Raman spectroscopies do not give extensive information about urine, they could become important tools for the routine clinical diagnostics of kidney problems, due to rapidness and low-cost. Thus, it is important to review all the applicable techniques and methods related to urine analysis. In this review, a brief overview of each technique’s principle is introduced. Where applicable, research papers about protein determination in urine are summarized with the main figures of merits, such as the limit of detection, the detectable range, recovery and accuracy, when available.

Urine is a readily available liquid for the medical diagnosis of patients. Urine tests are noninvasive procedures that involve no pain or discomfort for patients to determine problems with kidney function since extensive medical research showed that patients, with high protein concentration in urine, have various kinds of illnesses of the kidney, referred to as proteinuria [1] . Thus the precise and simple determination of urinary concentrations of total protein is important for diagnostic purposes. Since urine is a complex matrix, it poses challenges for the analytical determination of proteins and other constituents. Among the reasons that make urine challenging for researchers to analyze are: urine matrix is complex, it consists of various inorganic and organic compounds, from low-molar mass molecules to polymers; urine could contain cells, such as blood cells, or bacteria, which changes the composition of urine in time rapidly; an analytical method for diagnosis of proteinuria should cover protein presence in urine in a wide range from 0.01 mg/ml to 10 mg/ml. Also, it is important to note that the concentration of protein in urine taken from patients can vary widely depending on dieting, exercising, and time of the day a patient urinated. It is widely accepted that analysis of urea taken throughout a day is the best representation of any illnesses in the human body if any. The so-calledthe urine 24-hour volume test, that measures the amount of urine the human body produces in a day.

Another option is to determine creatinine to protein ratio in urea since creatinine to protein ratio is positively correlated with 24-hour volume test for quantifying proteinuria [2, 3] . The noninvasive collection of samples and wide range of diagnostic targets found in urine makes urinalysis well suited for point-of-care (PoC) monitoring applications, in which testing is done at the time and place of patient care [4] . Table 1 shows constituents in urine taken from the urine 24-hour volume test. Table 1 . Composition of urine (averages of selected components in 24-hour collection test) [5] .

Average weight (mg) Table 1 . Composition of urine (averages of selected components in 24-hour collection test) [5] .

Albumin 90.0 In healthy individuals, Tamm -Horsfall protein (also known as uromodulin) is the most abundant protein in urine (50%), followed by albumin (20%) and immunoglobulin (5%) [6] . Other constituents in the urine of either sick or healthy patients include, but not limited to: glucose; low abundant proteins, such as bilirubin, and urobilinogen; cells, such as Erythrocyte, Leukocyte, and other cells; bacteria [7] .

The urine of a healthy individual contains up to 150 mg of protein in total measured throughout a day, of which approximately 20 mg is albumin (human serum albumin) [1] . Albumin excretion of 30

to 300 mg a day, which is called microalbuminuria, is an early and sensitive marker of diabetic nephropathy [8] , cardiovascular and renal disease [9] . The 15-30 mg/L albumin concentration is a critical value that could indicate kidney problems when it is repeatedly exceeded [10] . Though one of the main proteins in urine is albumin, there are thousands of other types of proteins. Furthermore, the removal of albumin from the urine helps to identify the low abundant proteins [11] . Marimuthu et al. by

using high-resolution Fourier transform mass spectrometry were able to identify 1823 proteins in the urine of healthy subjects [12] . Using mass spectroscopy and sub-fractionating normal urine by successive steps (vesicle separation, CPLL, and solvent treatments) Santucci et al. were able to identify 3429 individual proteins [13] . All those discovered proteins could be potential biomarkers for diseases.

In a review paper by Röthlisberger et al. it was stated that besides albumin, other proteins such as CD14, hh-FABP, BNP / NT-proBNP, NGAL, ORM1 are potential biomarkers of cardiovascular disease [14] . Another biomarker in human urine, Bence Jones protein (BJP), has an important diagnosis and prognosis value for multiple myeloma, cancer formed in a white blood cell called plasma cell [15] .

Other types of chemical substances in urine can be important biomarkers as well, for example, urine microRNAs have the potential to be a valid marker for bladder cancer detection [16] .

Proteinuria is the main clinical presentation of glomerular diseases (the glomerulus are complex capillary set that are located in the nephronsrenal cells) [17] . The glomerular diseases can be primarily when the disease caused by kidney diseases (such as glomerulonephritis), or secondary, when kidney glomerulus becomes target organ affected by different diseases such as diabetes and cardiovascular diseases, autoimmune and inflammatory disorders, amyloidosis and neoplasms, cancer J o u r n a l P r e -p r o o f and many other including genetic disorders [17, 18] . The levels of proteinuria, which usually measures in clinical settings, may vary depending on severity of disease and glomerular injury, despite any cause of disease. Any detectable proteinuria in the urine between 30 -300 mg/24h called albuminuria (or microalbuminuria), above the 300 mg/24h -proteinuria. Depending the amount of proteinuria they considered to named nephritic range proteinuria below 3.5 g/24h and nephrotic range proteinuria when protein loss excess the 3.5 g/24h (heavy proteinuria). The most of the cases, proteinuria caused by cardiovascular diseases limited with nephritic range proteinuria, whereas in renal diseases and cancer the proteinuria may reach nephrotic range proteinuria [19, 20] . Since urine is in direct contact with kidneys, analysis of urine is important for diagnosis of renal diseases, such as chronic kidney disease.

Several viral infections such as Epstein-Barr virus, hepatitis B and C viruses, herpes zoster, hantavirus, human immunodeficiency virus, dengue fever, COVID-19 and many others also may lead to proteinuria [21, 22] . The potential mechanisms of proteinuria are related to primary affect to glomerulus and/or secondary to autoimmune response to infection [23, 24] . Chronic kidney disease (CKD) is a major public health problem. Albuminuria, urinary sediment abnormality and other markers of kidney damage are criteria of CKD, according to international guidelines [25] . Fassett et al. summarized biomarkers for CKD in urinecystatin C, β-trace protein, NGAL, KIM-1, NAG and many others [26] . A more recent article on this topic is discussed in [27] .

In the United States, it was estimated in 2003 that 11% of the adult population (aged 20 or older) has chronic kidney disease [28] . In a review paper about the prevalence of chronic kidney disease (CKD) by Zhang et al., the authors studied relevant data across America, Europe, Asia, Australia [29] .

They reported that the median prevalence of CKD was 7.2% in persons aged 30 years or older. In persons aged 64 years or older prevalence of CKD varied from 23.4% to 35.8%. The authors concluded that worldwide, CKD is becoming a common disease in the general population. In a more recent paper, Wei [45] . They concluded that it is important to perform an investigation using non-invasive approaches, such as urine can provide, for the diagnostic of emerging viral diseases.

Urinalysis is an abbreviation for clinical urine tests for diagnostic purposes. A urinalysis (UA) is one of the most common methods of medical diagnosis. There are three basic components to urinalysis [46] :

• Gross/physical examination targets parameters that can be measured or quantified with the naked eye (or other senses), including volume, color, transparency, odor, and specific gravity.

• Microscopic examination. The numbers and types of cells and/or material such as urinary casts can yield a great detail of information and may suggest a specific diagnosis.

• Chemical examination of urine measures quantitatively and qualitatively for pH, blood, nitrite, protein, glucose, ketones, bilirubin, urobilinogen, ascorbic acid.

Conditions for storage of urine samples is an important consideration for chemical analysis, since undesired changes in unpreserved urine occur, such as a decrease of concentration of glucose due to consumption of it by cells or bacteria; a decrease of concentration of bilirubin due to photo-oxidation and etc [47] . The most common form of preservation of urine samples is refrigeration, also chemicals J o u r n a l P r e -p r o o f can be utilized too. Remer [55] .

To date, different diagnostics methods in clinical settings can be used to ascertain urinary protein and albumin. Urine dipstick test, technique with acidic buffer precipitation and immunoelectrophoresis are commonly used diagnostic methods for urinary total protein quantification [56] . The protein-specific dipstick and immunochemical techniques are the fastest and cheapest way to determine proteins in the urine for diagnostic purposes. Modern automated urine analyzers are based on similar principles and are widely used in clinical routine tests [57] . A urine analyzer is a device used in the clinical setting to perform automatic urine testing. The units can detect and quantify a number of analytes including bilirubin, protein, glucose, and red blood cells. Many models contain urine strip readers, a type of reflectance photometer that can process several hundred strips per hour. Some analyzers can perform up to 240 tests an hour. Prices of urine analysis are relatively low, but they J o u r n a l P r e -p r o o f depend on a location/country and they are available from the most clinical laboratories The highperformance liquid chromatography mostly used for scientific purpose due to its relatively high cost , though relatively inexpensive versions of mass spectrometers are now available [58] .

Prices of urine analysis are relatively low, but they depend on a location/country and they are available from the most clinical laboratories The high-performance liquid chromatography mostly used for scientific purpose due to its relatively high cost , though relatively inexpensive versions of mass spectrometers are now available [58] .

Several review papers are focused on urine proteomics. A review by Albalat et al. focuses on urinary proteins as potential biomarkers for mainly urine diseases, and capillary electrophoresis coupled mass spectroscopy as an instrumental method [33] . The review also gives information on biomarkers on non-renal diseases obtained from the urine. Kalantari et al. state that mass spectrometry as a detection technique is the most common [59] . The paper discusses technical aspects of urinary proteomics, proteomic technologies, and their advantage and disadvantages. As of 2015, several recent experiments are presented there which applied urinary proteome for biomarker discovery in renal diseases including diabetic nephropathy, immunoglobulin A (IgA) nephropathy, focal segmental glomerulosclerosis, lupus nephritis, membranous nephropathy, and acute kidney injury. Decramer et al. in their review paper focus on mass spectrometry-based urinary protein and peptide profiling [60] . The advantages and disadvantages of different mass spectroscopy methods are compared. Applications of urinary proteome analysis to urogenital and non-urogenital diseases are also discussed there.

This review, besides covering mass spectrometry, holds chapters discussing fluorescence spectroscopy, immunoassay, infrared, and Raman spectroscopy that was not given much attention compared from the reviews on urinalysis mentioned in the previous paragraph. This review also covers separation techniqueselectrophoresis and chromatography. For each technique, a brief overview of the technique's principle is introduced. Where applicable, research papers about protein determination in urine are summarized with the main figures of merits, such as the limit of detection, the detectable range, recovery, and accuracy. Particularly, electrophoresis, chromatography, immunoassay, and fluorescence spectroscopy chapters are followed by summary tables with analytical parameters taken from individual research papers. It should be noted that not all analytical parameters are given in every paper, for example, sometimes recovery is unknown, and thus is not included in the following tables. Since the volumes used for all techniques are 100 to 1000 times smaller than a typical urine probe available from one patient for analysis, usually 50-100 mL, urine volume is not introduced in the tables. For instance for proteomics study, a typical sample size for high performance liquid chromatography (HPLC) ranges from few microliters to 0.1 mL [61] . A commercial immunoassay test kits developed by Valle et al. require 10 μL of urine [62] . This review ends with a paragraph about urinary protein biomarkers. Most of those biomarkers were identified by mass spectrometry or immunoassay methods. A summary table of experimental papers on urinary protein biomarkers with related diseases or health conditions is given.

Electrophoresis is an electrokinetic process, with a long history of development, which separates charged particles (macro-molecules, such as proteins) in a fluid using a field of electrical charge [63] . Particles could be separated based on their charge or their mass. The electrophoretic method is a widely used method in a modern research laboratory, but not so much in a clinical laboratory. Electrophoresis is commonly used for proteins, peptides and nucleic acid analysis [64] . It could be applied as a qualitative or quantitative analytical technique on its own if a reference substance is given, or it could be used as a separation technique for a sample to be further examined by other techniques, such as mass spectroscopy, fluorescence spectroscopy and etc.

Aguzzie et al. successfully used the electrophoretic technique to detect Bence-Jones protein (BJP) in human urine with the limit of detection LOD of 1 mg/L to BJP, and LOD of 3 mg/L to other types of protein [65] . These results were obtained by immunofixation using Dako antisera on cellulose nitrate followed by further staining with gold. The authors claim that their technique does not require particular skill and is cheap. Such low levels of LOD allowed them to use unconcentrated urine samples, thereby eliminating the problem of the loss of low molecular weight proteins. Usually, though it is often necessary to concentrate the urine before electrophoresis because of the low urinary protein level [6] . This is a time-consuming process. Machii et al. used cellulose acetate membrane electrophoresis followed by colloidal silver staining on the urine of healthy subjects with success [6] .

However, when they used silver staining on an agarose gel, urinary protein in healthy subjects was not detected. They estimated that urine has to be concentrated 10 times, furthermore, the pattern of the urinary fraction will change after the concentration procedure is conducted. Giovannoli et al. developed a noncompetitive capillary electrophoresis immunoassay with laser-induced fluorescence detection was used to determine human serum albumin (HSA) in buffer and urine [66] . The injection of an excess of labeled antibody off-line incubated with the analyte allows the surface capture of the free antibody and consequently the immunocomplex detection. In buffer, authors were able to achieve the limit of detection LOD of 0.60 mg/L, with the quantitative range up to 6.6 mg/L. Recovery in the urine matrix was dependent upon sample dilution and HSA added and was as low as 91±8%.

One of the variations of the electrophoretic method is called gel electrophoresis, where a gel is used as an anticonvective medium. Particularly polyacrylamide gel PAGE is used for separating [69] . The use of these preconcentration techniques makes it possible to increase sensitivity in the determination of analytes by a factor of 100 or higher. In another work by Bessonova et al., they found that the lowest LOD was obtained by the LVSS approach, and it was 15 mg/L with UV-detection [70] . [68] . A gel column is utilized to achieve electrophoretic separation of proteins, analogous to SDS-PAGE, which are then eluted into the liquid-phase for manual collection. The fractionation can then be visualized by running a portion of the fractions on a SDS-PAGE gel. Reprinted from [95] .

Electrophoresis is often used to be followed by another method, such as mass spectroscopy. 

Another widely used separation technique in chemistry is chromatography. In chromatography, a sample is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate due to differential affinities (strength of adhesion, as an example) of the various components of the analyte towards the stationary and mobile phase results. Then resulting constituents can be further analyzed by various techniques, such as mass spectroscopy, UVspectroscopy, fluorescence detection, etc. Depending on the mobile phase methods in chromatography could be classified as gas-chromatography (GC) [74] , and liquid-chromatography (LC) [75] . Typically, proteomic sample size ranges from few microliters to 0. [76] . Particularly, they used 8-anilino-1-naphthalenesulfonic acid as a fluorophore to bind HSA. The sample size was 1 ml for urine, and the retention time was around 20 minutes. A linear relationship was observed between fluorescence peak and the amounts of HSA in both plasma and urine, as for the latter matrix, a linear relationship was observed up to 400 mg/L, with the correlation coefficient being 0.998. The LOD for HSA was 0.2 mg/L, while the recovery was 94.5±3.8%. The same authors also compared their method with the immunological nephelometric method and obtained the correlation coefficient of 0.990 [77] . Often researchers compare their method with the immunoassay technique that is used in clinical practice. Brinkman et al. in their experiments using the immunonephelometric approach and HPLC determined that HPLC reveals higher values of HSA, especially in the lower concentration range, resulting in a higher prevalence of microalbuminuria [78] . [85] . AQPs regulate numerous downstream effector signaling molecules that promote cancer development and progression [86] . In numerous cancer types, AQP expression has shown a correlation with tumor stage and prognosis. Linearity is observed within the concentration range 0.5·10 -3 -50·10 -3 mg/L, intra and inter-assay precision ranged from 9 to 35% at the lower limit of quantification (LLOQ), and accuracy from 94 to 114%.

Overall, due to complex composition of biofluids, including the urine, liquid chromatog-raphy in particular HPLC, which is frequently coupled with MS for detection, remains a common method in bioanalytical laboratories to separate complex mixtures. A review by Novakova et al. states that in order to decrease time for an analysis without compromising resolution and separation efficiency, three main approaches in HPLC existthe use of monolith columns, LC at high pressures and temperatures, and HPLC at ultra-high pressures [87] . By employing those approaches, time for analysis of biofluids could be done within few minutes up to half an hourhe Table 2 below shows a summary table of quantitative methods for application of electrophoresis and chromatography for protein determination in the human urine matrix. Table 2 . Summary table of quantitative methods for application of electrophoresis and chromatography for protein determination in the human urine matrix (unless otherwise stated in the method section). CV (coefficient of variation) parameters are taken from maximum readings, for Recovery -the range is given. J o u r n a l P r e -p r o o f

Mass spectrometry is a powerful analytical technique that measures the mass-to-charge ratio of ions. Firstly, molecules in a sample must be ionized, which causes a large molecule to fragment into smaller ions; then those ions are separated by a magnetic field depending on their mass to charge ratio.

The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-tocharge ratio. Usually, mass spectrometry is utilized after pre-treatment of a sample by electrophoresis or chromatography.

Mass spectrometry is a widely used technique for proteomics studies. The proteome is the entire set of proteins that is produced or modified by an organism. Research on urine proteomics is important for the identification purposes of reliable biomarkers that help the diagnosis of disease [90] . In a review by Kalantari et al. on urine proteomics is stated that common techniques for urinary proteome analysis are two-dimensional gel electrophoresis followed by mass spectrometry (2DE-MS), liquid chromatography coupled to mass spectrometry (LC-MS), surface-enhanced laser desorption/ionization coupled to mass spectrometry (SELDI-TOF), and capillary electrophoresis coupled to mass spectrometry (CE-MS) [59] . By using mass spectroscopy protein constituents of urine were identified.

Also reviews by Albalat et al. and Decramer et al. focus on mass spectrometry-based urinary protein and peptide profiling [33, 60] . The advantages and disadvantages of different mass spectrometry methods are compared. As was mentioned in the introductory part of this review, Marimuthu et al. by

using high-resolution Fourier transform mass spectrometry were able to identify 1823 proteins in the urine of healthy subjects [12] . Santuccie et al. by using mass spectrometry and sub-fractionating normal urine by successive steps (vesicle separation, CPLL, and solvent treatments) were able to identify 3429 individual proteins [13] .

Two ionization techniques in mass spectrometry are commonly used for protein studies: electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) [91] . In the ESI approach, ions are produced in an electrospray in which a high voltage is applied to a liquid to create an aerosol. ESI is different from other ionization processes (e.g. MALDI) since it may produce multiplecharged ions, effectively extending the mass range of the analyzer to accommodate the kDa-MDa orders of the magnitude observed in proteins and their associated polypeptide fragments [92] . In MALDI, a sample is dispersed in a certain solid matrix (e.g. aromatic compounds), which then irradiated by the laser that causes electronic excitation of molecules in the sample matrix [93] . Both J o u r n a l P r e -p r o o f methods ESI and MALDI produce relatively high molecular ions due to mild fragmentation of those approaches. It should be noted a specific method of MS applied to biomolecules, which is called tandem mass spectrometry, also known as MS/MS. In MS/MS two or more mass analyzers are coupled together using an additional reaction step to increase their abilities to analyze samples. In general, protein identification in mass spectroscopy is performed using either bottom-up or top-down approaches. Bottom-up protein identification methods rely on enzymatic digestion to break down proteins into smaller peptides that are easier to ionize and fragment, resulting in higher sequence coverage [94] . However, in top-down methods, intact proteins are injected into the mass spectrometer and subjected to fragmentation without pre-treatment [95] . Aside from better tracking of protein modifications, an advantage of top-down protein sequencing is that it complements imaging experiments by enabling mass measurement of the intact protein that relates more directly to the MALDI IMS generated signals [96] .

Analysis of a mass-spectrum, a plot of intensity as a function of the mass-to-charge ratio, could be a challenge itself, especially for big molecules like proteins, since they can be divided in many fragments resulting in a complex spectrum. Usually, the mass-spectrum is compared by software with the existing database. Several computationally feasible approaches have emerged for performing protein inference from such data [97] . Recovery was between 97% and 108% with an average of 103%. In conclusion, quantitative mass spectroscopy assays for peptides and proteins are difficult to implement, and the future will see standardization of MS validation and quality control requirements.

Overall, mass spectrometry coupled with liquid chromatography is a powerful tool for the discovery and the analysis of biomarkers of diseases from biofluid samples, which includes proteomics study of urine as well [104] . Many of urinary proteome biomarkers were discovered by this technique.

However, quantitative protein determination represents a challenge by following this approach [105] .

Thus, mass spectroscopy could be used for biomarker discovery but not for clinical applications [60] .

Another consideration for an analytical technique is the cost of analysis. Practical usage of LC-MS is limited by factors such as instrument size, cost, ease of operation, though, low cost compact mass spectroscopy detectors coupled with cinematographic separation are being developed, their costs equal to around $50000 [58] .

Immunoassay is a widely used technique in clinical and research laboratories to determine qualitatively and quantitatively macromolecules (usually proteins) generated from organisms [106] .

Immunoassay is based on a reaction between an antigen and an antibody, which is found in the adaptive immune system of vertebrates, that includes humans [107] . The reaction is carried out in-vitro (not in a living organism), and the result of it is the formation of a complex antibody-antigen formed by spatial complementary, like lock and key, thus such interactions are highly selective. Antibody-antigen complexes are bounded by weak forces such as electrostatic forces, hydrogen bonds, hydrophobic interactions, and van der Waals forces; which means that such bindings are reversible. The specific piece of the antigen to which an antibody binds is called the epitope. Usually, in analytical papers, the analyte is the antigen while the reagent is the antibody. Immunoassay methods have been widely used in many areas of pharmaceutical analysis such as diagnosis of diseases, therapeutic drug monitoring, clinical pharmacokinetics, and bioequivalence studies in drug discovery and pharmaceutical industries J o u r n a l P r e -p r o o f [108] . Conventional immuno-chemical based urinary albumin assays for diabetic patients as well as nondiabetic patients with renal disease, including those who may suffer hypertension and cardiovascular disease, now amount to 100 million assays per year worldwide as of 2004 [79] . The widespread immunoassay methods in the pharmaceutical analysis is attributed to their inherent specificity, high-throughput, and high sensitivity for the analysis of a wide range of analytes in biological samples as well the relatively low cost of the instruments, tools, or the reagents [109] .

Immunoassay experiments could be classified into types -competitive and non-competitive [106, 109] . [109] .

Based on signal detection immunoassays could be classified as follows:

• Radioimmunoassay RIA. Detection is based on radioactive labeling of antigen with unstable isotopes such as I-125, C-14, H-3.

• Enzyme immunoassay EIA with sub-categories: ELISA -Enzyme-linked immunosorbent assay; EMIT -Enzyme-multiplied immunoassay technique; MEIA -Microparticle enzyme immunoassay.

• Fluoroimmunoassay FIA

• Chemiluminescence immunoassay CLIA

• Immunonephelometry IN. Detection is based on an antigen-antibody complex scattering light.

• Immunoturbidimetry IT. Detection is based on an antigen-antibody complex blocking light thus increasing the turbidity of the sample.

As it was mentioned in the introductory part, the concentration of albumin higher than 2 mg/L could be indicative of potential kidney diseases [10] . Subsequently for many immunoassays methods for albumin determination in urine the limit of detection (LOD) of magnitude around 1 mg/L would be enough for clinical purposes, thus priority should be given to the parameters such as specificity, accuracy, costs, etc. However for low abundant proteins achieving lower LOD is important [110] . In Table  3 . Table 3 . Summary table of quantitative methods for application of immunoassay for protein determination in the human urine matrix (unless otherwise stated in the method section). CV (coefficient of variation ) parameters are taken from maximum readings, for Recovery -the range is given. J o u r n a l P r e -p r o o f

Fluorescence is a luminescence phenomenon in which a compound emits light after absorption of electromagnetic irradiation [133] . In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. Often in chemical analysis, a sample is irradiated in UV or visible range, and as a result, fluorescence occurs in the visible and near-infrared region respectively. Fluorescent materials cease to glow nearly immediately when the radiation source stops, unlike phosphorescent materials, which continue to emit light for some time after. Fluorescence spectroscopy is one of the most common techniques with applications in geology, chemistry, medicine, and astronomy [133] . Because of the sensitivity that the method affords, fluorescent molecule concentrations as low as 1 part per trillion can be measured [134] . Fluorescence spectroscopy can be used on its own, and also it is often used in conjunction with high-performance liquid chromatography as a detector. Often fluorescence detection is used in immunoassays to either label antigen or antibody.

Usually, analyte molecules do not emit strong luminescence for their analytical detection. Hence fluorescence quenching aptasensor [132] . When albumin was added to the complex GO with the fluorescence-labeled aptamer, the aptamer detached from the complex to bind albumin, which resulted by an increase in fluorescence intensity, as shown in Figure 4 . The limit of detection was 0.05 mg/L and the detection range is 0.1-14.0 mg/L. Other quantitative works using the fluorescence spectroscopy are summarized in Table 4 .

J o u r n a l P r e -p r o o f Figure 4 : Schematic of graphene oxide-mediated fluorescence quenching aptasensor for the detection of albuminuria in urine and HSA in human serum. When albumin was added to the complex GO with the fluorescence-labeled aptamer, the aptamer detached from the complex to bind albumin, which resulted by an increase in fluorescence intensity. Reprinted from [132] . Table 4 . Summary table of quantitative methods for application of fluorescence spectroscopy for protein determination in the human urine matrix (unless otherwise stated in the method section). CV(coefficient of variation) parameters are taken from maximum readings, for Recovery -the range is given. J o u r n a l P r e -p r o o f

The absorption of infrared radiation IR excites vibrational transitions of molecules [148] . The infrared spectral region covers wavelengths from 780 nm to 1000 μm which can be further subdivided into the near-infrared region from 780 nm to 2500 nm, the mid-infrared region from 2500 nm to 50 μm and the far-infrared region from 50 μm to 1000 μm. Since vibrational frequency and probability of absorption depend on the strength and polarity of the vibrating bonds, they are influenced by intra-and intermolecular effects [149] . The approximate position of an infrared absorption band is determined by the vibrating masses and the type of bond (single, double, triple), the exact position by electronwithdrawing or donating effects of the intra-and intermolecular environment and by coupling with other vibrations. Information that can be derived from the infrared spectrum:

• Chemical structure of the vibrating group

• Chemical properties of neighbouring groups in a molecule

• Redox state

• Bond parameters

• Hydrogen bonding

• Electric fields

• Conformational freedom Basically, all polar bonds contribute to the infrared absorption so there is no need to specifically label biomolecules to detect them. However, the infrared spectrum of biomolecules is challenging to analyze since they are complex with many overlapping bands. Certain mathematical and statistical procedures have to be utilized to analyze the resulting spectrum. The IR spectroscopy is often used to identify organic structures because functional groups give rise to characteristic bands both in terms of intensity and position (frequency). The IR spectra of organic molecules are often interpreted as having two regions: functional group region (>1500 cm -1 =6.7 μm), and fingerprint region (<1500 cm -1 ). In the fingerprint region, there are many troughs that form an intricate pattern that can be used as a fingerprint to determine the compound. Modern infrared spectrometers are usually Fourier transform infrared (FTIR) spectrometers [149] . The FTIR spectrometer works differently than the dispersive spectrometer where a monochromatic light beam is absorbed by a sample. This technique shines a beam containing many frequencies of light at once and measures how much of that beam is absorbed by the sample.

Next, the beam is modified to contain a different combination of frequencies, giving a second data point. This process is rapidly repeated many times over a short timespan. Afterward, a computer takes Another analytical technique that relies on molecular vibrations is Raman spectroscopy [150] .

Raman spectroscopy is a powerful technique to determine vibrational modes of molecules to provide a structural fingerprint, like infrared spectroscopy, by which molecules can be identified. Infrared Raman spectra for biomolecules is hard to analyze due to overlapping bands, the same issue as in the IR spectroscopy. Certain mathematical procedures must be employed on Raman spectra to analyze a sample.

Despite difficulties in the analysis of large biomolecules such as proteins, the IR, and Raman spectroscopy make their advances in this regard. For the protein analysis with IR, the MIR region plays an important role since it includes bands that predominantly arise from three conformationally sensitive vibrations arising from the peptide backbone-namely, amide I, amide II, and amide III [151] . Amide group vibrations of the backbone have attracted much attention in protein IR spectroscopy, as they are native to all proteins and inform on secondary conformation and solvation. Such bands include the amide I region, between 1600 and 1700 cm-1, primarily resulting from C=O stretching vibration, the amide II band, related to CN stretch and NH in-plane bending, the amide III vibration, associated with CN stretching, NH bending, and CO in-plane bending, and, finally, the amide A (NH stretch) band.

Absorption Spectroscopy (SEIRAS) [152] , and Surface-Enhanced Raman spectroscopy SERS [153] [154] [155] improve sensitivity to overcome this problem. Both SEIRAS and SERS utilize the surface plasmon effect from the interaction of light with metallic nanoparticles. SERS has an enhancement factor (of the order of 10 6 -10 12 ) compared to the traditional Raman signal [156] . For SEIRA, the surface-enhancement is comparatively modest ( 10 1 -10 3 ) [157] . Although the enhancement factor of SEIRAS is smaller than that of SERS, the cross-section for IR absorption is several orders of magnitude higher than the corresponding Raman cross-section. Despite the modest enhancement factor, the SEIRAS technique may be sufficient for many applications [152] . [160] . Pezanniti et al. used a polynomial based spectral smoothing method to the urine matrix [161] . In general, all the mentioned works requires no specific reagents, they are fast and are able to quantify multiple analytes with one spectrum.

Premasiri et al. investigated urine by Raman spectroscopy [5] . shown in Figure 5 [164] . The authors used the set of mathematical procedures on spectra which included spectral processing (e.g., truncation, baselining, and vector normalization); principal component analysis (PCA); statistical analyses (ANOVA and pairwise comparisons); discriminant analysis of principal components (DAPC); and testing DAPC models using a leave-one-out build/test validation procedure. Their approach was able to identify "unknown" urine specimens as from PD patients or healthy human volunteers with better than 96% accuracy (with better than 97% sensitivity and 94% specificity). 1. for albumin -peaks at 1208 cm −1 and 1370 cm −1 are attributed to SO 2 symmetric stretch and CH bond deformation of albumin.

2. for creatinine -the Raman signature peaks are observed at 888 cm −1 , 958 cm -1 , and 1444 cm-1 which could be due to C=O stretching, C-C stretching and CH3 anisometric deformation respectively.

3. for urea -a very strong Raman peak at 1018 cm −1 which corresponds to C-N stretching mode.

Throughout is important consideration for laboratory tests. Zhu et al. performed quantitative SERS detection of creatinine in urine, and then compared with a clinically validated enzymatic "creatinine kit" [168] . They wrote that SERS detection process could be completed within 2 min compared with 11 min for the creatinine kit, indicating the practicality of the quantitative SERS technique as high-throughput platform for relevant clinical and forensic analysis.

Human urine contains thousands of individual proteins [12, 13] . Excessive presence of some of them are associated with certain diseases related not only to renal diseases, but also to cancer, diabetes, and infections. Those molecules that are associated with a certain biological condition are called biomarkers. Most of those biomarkers in human urine were identified by comparing patients with healthy individuals by the means of chromatography or electrophoretic separation followed by mass spectroscopy, or by the immunoassay. Literature review of urinary protein biomarkers with related diseases is summarized in Table 5 , corresponding diagnostic sensitivity and diagnostic specificity are given [169] . Some of the listed research papers used Western blot as a technique [170] . Due to the usage of antibodies, this technique was counted as the immunoassay in Table 5 . Most of the methods mentioned followed established procedures. To get a general idea of those experimental methods, refer

to the previous paragraphs about separation and detection techniques, and Tables 2-4 .

Below are some observations and conclusions regarding Table 5 about the content of the listed research papers:

• Urinary protein analysis are useful for not only renal diseases. Urine proteomics can be used for detection of at least 15 conditions, including 8 kinds of cancer; 5 kinds of renal complications; and at least 3 different infections. The list of health complications that can be diagnosed from J o u r n a l P r e -p r o o f urine analysis is bigger if non-protein substances are included, such as peptides, nucleic acids, low molar mass metabolites, and etc; that can be found in reviews [14, 26, 27, 36] .

• New potential urinary protein biomarkers are found primarily by mass spectrometry nowadays. However, further extensive validation on clinically representative population is required. Often it is achieved through the immunoassay, if the appropriate method is developed with a sufficient analytical sensitivity, since the immunoassay methods are expressive, low-cost and quantitative compared to the chromatography coupled with mass spectrometry.

• In total 34 references are included in Table 5 ; 12 of them utilized mass spectrometry , 24 utilized the immunoassay, and 1 paper -fluorescence spectroscopy.

• Authors of these research papers utilized the mass spectrometry methods with further validation by the immunoassay - [171] [172] [173] [174] .

• All of the mentioned work (except [129] ) in the immunoassay followed manufacturer's protocol, or if a custom immunoassay was used, other established procedure from their own or previous works.

• ELISA-based immunoassay was the most popular method accounting for 13 references.

• Diagnostic sensitivity and specificity increased if a panel of several biomarkers was considered simultaneously. Though, the panel should be carefully chosen as not all of the components provide increase in diagnostic sensitivity and specificity. 

Urine is a readily available liquid for the medical diagnosis of patients. Urine tests are noninvasive procedures that involve no pain or discomfort for patients to determine problems with kidney function since extensive medical research showed that patients, with high protein concentration in urine, have various kinds of illnesses of the kidney, referred to as proteinuria [1] . Thus the precise and simple determination of urinary concentrations of total protein is important for diagnostic purposes. In this review, we focused on instrumental determination of abundant proteins in urine by electrophoresis, chromatography, mass spectrometry, immunoassay, fluorescence, IR, and Raman spectroscopy. The mentioned techniques are not mutually exclusive, for example, high performance liquid chromatography is usually a separation step followed by mass spectrometry, another example is labelling with fluorophores in immunoassay. Each technique has its own strength to analyze urine for specific information, hence, it is hard to compare them directly, and to find a common denominator for comparison. Overall, number of cited experimental papers that were covered by this review in Tables 2-5 : electrophoresis and chromatography -22 in total and 8 related to HSA detection; mass spectrometry -17 in total and 5 related to HSA; immunoassay -47 in total and 17 related to HSA;

fluorescence spectroscopy -22 in total and 19 related to HSA; IR and Raman spectroscopy -10 in total; all topics -101 papers. Thus, almost half of the cited literature by the means of immunoassays, which is in a good correlation that immunoassays are widespread in clinics and science.

The limit of detection is important parameter for evaluating an analytical method. In order to develop a method, the limit of detection should take into account which amount of analyte has clinical significance. The 15-30 mg/L albumin concentration is a critical value that could indicate kidney problems when it is repeatedly exceeded [10] . For the corresponding articles discussed above, the limit of detection for electrophoresis, chromatography, mass spectroscopy, immunoassay, fluorescence exceeded 2 mg/L for albumin, which is enough for the clinical purpose. For some methods, explained in [117, 119, 122, 123, 127, 132, 143] , the limit of detection does not exceed 0.1 mg/L for albumin in the urine matrix. For low abundant proteins the limit of detection must be obviously much lower. For example, in the article for Aquaporin-2 determination by Jaffuel et al. the limit of detection was 5·10 -4 mg/L [85] . In another work by Zhao et al., the LOD for Nuclear matrix protein 22 was 1.7·10 -6 mg/L [110] . For the IR and Raman spectroscopy methods the LOD might not be so relevant factor. The IR and Raman methods is better characterized by how accurate a particular method can distinguish whether a spectra belongs to a healthy subject or not, as evidenced from works [158, 164] .

J o u r n a l P r e -p r o o f Electrophoresis and chromatography are powerful separation techniques which are accompanied by further detection, usually UV-vis spectrometry or fluorescence. Since the human urine matrix is very complex consisting of inorganic and organic compounds, those techniques are useful for urine analysis [7] . Though certain methods might require preconcentration techniques to enhance sensitivity [69] . High-performance liquid chromatography with mass spectroscopy detection is a particularly powerful tool for proteomics studies, as it was able to identify 1823 proteins in urine [12] .

mass spectroscopy in itself does not allow for quantitative analysis due to the different physicochemical properties of different peptides and proteins [98] [99] [100] . However quantification in MS is possible if an internal standard is used, for such there are approaches based on labeling with stable isotopes (such as N-15, O-18), involving artificial labeling of peptides and proteins, as in work by Singh et al., specifically in the urine matrix [81] . The immunoassay is widely used technique both in clinical practice and in research papers for urine analysis. Though different immunoassay methods can overestimate or underestimate an analyte concentration [79] . They can hurt patients due to inaccurate results [112] . The IR and Raman spectroscopy have advantage of easy preparation of samples to analyze. They require no specific reagents, or no reagents at all, no preconcentration or dilution, as evidenced from [158, 162, 165] . They are rapid. On the other hand, due to overlapping bands of large biomolecules, and the complexity of urine components -hard to analyze their spectra that require mathematical analysis.

Overall, there is no "silver bullet" modern quantification method for the analysis of proteins in urine that has been reported and validated so far. However, at least for the routine clinical diagnostics of kidney problems, applications of relatively direct methods (such as SERS or IR) spectroscopy may expand as instrumentation, substrates (at least SERS substrates), software and computing power for data analysis (peak deconvolution) become progressively less expensive.

J o u r n a l P r e -p r o o f [68] . A gel column is utilized to achieve electrophoretic separation of proteins, analogous to SDS-PAGE, which are then eluted into the liquid-phase for manual collection. The fractionation can then be visualized by running a portion of the fractions on a SDS-PAGE gel. Reprinted from [95] . 

Primary Care Approach to Proteinuria

Correlation of urine protein/creatinine ratios to 24-h urinary protein for quantitating proteinuria in children

Diagnostic utility of protein to creatinine ratio (P/C ratio) in spot urine sample within routine clinical practice

Review-Point-of-Care Urinalysis with Emerging Sensing and Imaging Technologies

Urine analysis by laser Raman spectroscopy

Urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining

Urinalysis: A comprehensive review

Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus

Update on microalbuminuria as a biomarker in renal and cardiovascular disease

An overview of urinary proteomics applications in human diseases

A Comprehensive Map of the Human Urinary Proteome

From hundreds to thousands: Widening the normal human Urinome (1)

Urine protein biomarkers for detection of cardiovascular disease and their use for the clinic

History of Multiple Myeloma

Urine miRNA as a potential biomarker for bladder cancer detection -a meta-analysis

Proteinuria in adults: a diagnostic approach

Proteinuria: potential causes and approach to evaluation

Classification of renal proteinuria: a simple algorithm

Pathophysiology of proteinuria

Proteinuria in children infected with the human immunodeficiency virus

Proteinuria in Children: Evaluation and Differential Diagnosis

Understanding the Mechanisms of Proteinuria: Therapeutic Implications

Chronic Kidney Disease

Blood and urine biomarkers in chronic kidney disease: An update

Prevalence of chronic kidney disease and decreased kidney function in the adult US population: Third national health and nutrition examination survey

Prevalence of chronic kidney disease in population-based studies: Systematic review

Prevalence of kidney damage in Chinese elderly: a large-scale population-based study

Chapter 3: Morbidity and Mortality in Patients With CKD

Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study

Urine proteomics in clinical applications: technologies, principal considerations and clinical implementation

Discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis

Virus Bioresistor (VBR) for Detection of Bladder Cancer Marker DJ-1 in Urine at 10 pM in One Minute

Update on urine as a biomarker in cancer: a necessary review of an old story

Urinary Proteomic Biomarkers in Coronary Artery Disease

Urinary Proteomics in Diabetes and CKD

The Potential of Albuminuria as a Biomarker of Diabetic Complications

An immunoassay cassette with a handheld reader for HIV urine testing in point-of-care diagnostics

Analysis of viruses present in urine from patients with interstitial cystitis

Development of Universal and Lineage-Specific Primer Sets for Rapid Detection of the Zika Virus (ZIKV) in Blood and Urine Samples Using One-Step Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay

Detection and Phylogenetic Analysis of Human Papilloma Virus in Urine from a Sample of Iraqi Women with Vaginal Discharge

Find the right sample: A study on the versatility of saliva and urine samples for the diagnosis of emerging viruses

Urinalysis: A Review of Methods and Procedures

Fundamentals of Urine and Body Fluid Analysis

Long-term urine biobanking: Storage stability of clinical chemical parameters under moderate freezing conditions without use of preservatives

Free light chains nephelometric assay: human urine stability in different storage conditions

Effect of common storage temperatures and container types on urine protein : creatinine ratios in urine samples of proteinuric dogs

Comparison of various methods for the determination of total protein in urine

Protein Measurement with the Folin Phenol Reagent

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Measurement of protein using bicinchoninic acid

Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer

Assessment of proteinuria

Semiquantitative, fully automated urine test strip analysis

The emergence of low-cost compact mass spectrometry detectors for chromatographic analysis

Human Urine Proteomics: Analytical Techniques and Clinical Applications in Renal Diseases

Urine in Clinical Proteomics

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column

An enzyme immunoassay for determining albumin in human urine samples using an ultra-microanalytical system

Electrophoresis: The march of pennies, the march of dimes

Recent developments in capillary and microchip electroseparations of peptides

High-Sensitivity Electrophoretic Method for the Detection of Bence Jones Protein and for the Study of Proteinuria in Unconcentrated Urines

A novel approach for a non competitive capillary electrophoresis immunoassay with laser-induced fluorescence detection for the determination of human serum albumin

Urine protein quantification in stacking gel by SDS-PAGE

Gel-Eluted Liquid Fraction Entrapment Electrophoresis: An Electrophoretic Method for Broad Molecular Weight Range Proteome Separation

Preconcentration techniques in capillary electrophoresis

Electrophoretic determination of albumin in urine using on-line concentration techniques

Determination of peptides and proteins in human urine with capillary electrophoresis-mass spectrometry, a suitable tool for the establishment of new diagnostic markers

Mapping and Identification of the Urine Proteome of Prostate Cancer Patients by 2D PAGE/MS

Cellulose Acetate Membrane Electrophoresis Based Urinary Proteomics for the Identification of Characteristic Proteins: CAME-Based Urinary Proteomics

Fast gas chromatography-mass spectrometry: A review of the last decade

High-performance liquid chromatography combined with electron ionization mass spectrometry: A review

High-Performance Liquid Chromatographic Determination of Human Serum Albumin in Plasma and Urine by Post-column Fluorescence Enhancement Detection Using 8-Anilino-1-naphthalenesulfonic Acid

Manual Immunonephelometric Assay of Proteins, with Use of Polymer Enhancement

Which method for quantifying urinary albumin excretion gives what outcome? A comparison of immunonephelometry with HPLC

Differences in urinary albumin detected by four immunoassays and high-performance liquid chromatography

Performance Characteristics of an HPLC Assay for Urinary Albumin

A Liquid Chromatography-Mass Spectrometry Method for the Quantification of Urinary Albumin using a Novel 15N-Isotopically Labeled Albumin Internal Standard

Principles of Micellar Electrokinetic Capillary Chromatography Applied in Pharmaceutical Analysis

Optimization and Validation of a Capillary MEKC Method for Determination of Proteins in Urine

Field-amplified sample injection for the determination of albumin and transferrin in human urines by MEKC

Optimization of liquid chromatography-multiple reaction monitoring cubed mass spectrometry assay for protein quantification: Application to aquaporin-2 water channel in human urine

Water transport proteins-aquaporins (AQPs) in cancer biology

A review of current trends and advances in modern bio-analytical methods: Chromatography and sample preparation

Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine

Detection of urine protein by a paper-based analytical device enhanced with ion concentration polarization effect, Microfluid Nanofluid

Proteomics approach and techniques in identification of reliable biomarkers for diseases

Interpretation of Mass Spectra

Electrospray Ionisation Mass Spectrometry: Principles and Clinical Applications

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Biopolymers

Top Down proteomics: Facts and perspectives

Protein identification strategies in MALDI imaging mass spectrometry: a brief review

A review of statistical methods for protein identification using tandem mass spectrometry

Quantitative Mass Spectrometry-Based Proteomics: An Overview

Quantitative mass spectrometry: an overview

Clinical peptide and protein quantification by mass spectrometry (MS)

Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers

State of the Art for Measurement of Urine Albumin: Comparison of Routine Measurement Procedures to Isotope Dilution Tandem Mass Spectrometry

A reference system for urinary albumin: current status

Ultra high performance liquid chromatography as a tool for the discovery and the analysis of biomarkers of diseases: A review

Practical Considerations and Current Limitations in Quantitative Mass Spectrometry-based Proteomics

Immunoassays: Tools for Sensitive, Specific, and Accurate Test Results

Vertebrate and Invertebrate Respiratory Proteins, Lipoproteins and Other Body Fluid Proteins

Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective

Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances

A highly sensitive label-free electrochemical immunosensor based on AuNPs-PtNPs-MOFs for nuclear matrix protein 22 analysis in urine sample

Comparison between Immunoturbidimetry, Size-Exclusion Chromatography, and LC-MS to Quantify Urinary Albumin

The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry

A solid phase fluorescent immunoassay for the measurement of human urinary albumin

Urinary Albumin Measurement by Immunoturbidimetry

Laser immunonephelometry for routine quantification of urinary albumin excretion

Immunoturbidimetry of urinary albumin: prevention of adsorption of albumin

A rapid and sensitive method for estimating low concentrations of albumin in human urine

Time-resolved Fluorescence Resonance Energy Transfer Assay for Point-of-Care Testing of Urinary Albumin

Comparison and development of two different solid phase chemiluminescence ELISA for the determination of albumin in urine

A combination of magnetic permeability detection with nanometer-scaled superparamagnetic tracer and its application for one-step detection of human urinary albumin in undiluted urine

Electrochemical Immuno-Biosensor for the Rapid Determination of Nuclear Matrix Protein 22 (NMP22) antigen in Urine Samples by Co(III) Phthlocyanine/Fe3O4/Au Collide Coimmobilized Electrode

Resonance scattering detection of trace microalbumin using immunonanogold probe as the catalyst of Fehling reagent-glucose reaction

A quantum dot-based optical immunosensor for human serum albumin detection

A homogeneous fluorescent sensor for human serum albumin

Chemiluminescence lateral flow immunoassay cartridge with integrated amorphous silicon photosensors array for human serum albumin detection in urine samples

Competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles screen-printed electrodes: A potential rapid and noninvasive screening tool for early diagnosis of urinary tract carcinoma

Promptmas, Determination of Albumin in Urine by a Quartz Crystal Microbalance Label-Free Assay

A dual-label time-resolved fluorescence immunoassay for the simultaneous determination of cystatin C and β2-microglobulin in urine

Development of a fully automated chemiluminescence immunoassay for urine monomeric laminin-γ2 as a promising diagnostic tool of non-muscle invasive bladder cancer

Optical microchips based on high-affinity recombinant protein binders-Human serum albumin detection in urine

Electrochemical ELISA-based platform for bladder cancer protein biomarker detection in urine

Sensitive detection of albuminuria by graphene oxide-mediated fluorescence quenching aptasensor

Experimental methods in chemical engineering: Fluorescence emission spectroscopy

Fluorometric Assay Using Dimeric Dyes for Double-and Single-Stranded DNA and RNA with Picogram Sensitivity

Quantum dots versus organic dyes as fluorescent labels

Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging

Determination of human serum albumin using an intramolecular charge transfer fluorescence probe: 4′-Dimethylamino-2,5-dihydroxychalcone

Synchronous fluorescence determination of human serum albumin with methyl blue as a fluorescence probe

Synchronous fluorescence determination and molecular modeling of 5-Aminosalicylic acid (5-ASA) interacted with human serum albumin

Determination of Albumin Using CdS/SiO 2 Core/shell Nanoparticles as Fluorescence Probes

Protein determination using methylene blue in a synchronous fluorescence technique

Spectrofluorimetric Determination of Human Serum Albumin Using Terbium-Danofloxacin Probe

Determination of human albumin in serum and urine samples by constant energy synchronous fluorescence method

An NIR fluorescent probe of uric HSA for renal diseases warning

A Novel Detection Method of Human Serum Albumin Based on the Poly(Thymine)-Templated Copper Nanoparticles

A novel detection method of human serum albumin based on CuInZnS quantum dots-Co2+ sensing system

A red-NIR emissive probe for the selective detection of albumin in urine samples and live cells

Introduction

Infrared spectroscopy of proteins

Applications of Raman Spectroscopy in Agricultural Products and Food Analysis: A Review

Mid-infrared spectroscopy for protein analysis: potential and challenges

Recent applications of ATR FTIR spectroscopy and imaging to proteins

SERS Nanoparticles in Medicine: From Label-Free Detection to Spectroscopic Tagging

SERS: Materials, applications, and the future

Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering

Biochemical applications of surface-enhanced infrared absorption spectroscopy

Simultaneous determination of glucose, triglycerides, urea, cholesterol, albumin and total protein in human plasma by Fourier transform infrared spectroscopy: Direct clinical biochemistry without reagents

Quantification of albumin in urine using preconcentration and near-infrared diffuse reflectance spectroscopy

Near-infrared spectroscopic determination of serum total proteins, albumin, globulins, and urea

Preliminary investigation of near-infrared spectroscopic measurements of urea, creatinine, glucose, protein, and ketone in urine

Correlating the amount of urea, creatinine, and glucose in urine from patients with diabetes mellitus and hypertension with the risk of developing renal lesions by means of Raman spectroscopy and principal component analysis

Estimating the concentration of urea and creatinine in the human serum of normal and dialysis patients through Raman spectroscopy

Spectral characteristics of urine from patients with endstage kidney disease analyzed using Raman Chemometric Urinalysis (Rametrix)

Blu-ray DVD as SERS substrate for reliable detection of albumin, creatinine and urea in urine

Quantitative analysis of metabolites in urine using a highly precise, compact near-infrared Raman spectrometer

Development and validation of hybrid Brillouin-Raman spectroscopy for non-contact assessment of mechano-chemical properties of urine proteins as biomarkers of kidney diseases

Rapid and low-cost quantitative detection of creatinine in human urine with a portable Raman spectrometer

Sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings

Western Blot: Technique, Theory, and Trouble Shooting

Identification of a Three-Biomarker Panel in Urine for Early Detection of Pancreatic Adenocarcinoma

Proteomic analysis of urinary extracellular vesicles from high Gleason score prostate cancer

Exosomal proteins as prostate cancer biomarkers in urine: From mass spectrometry discovery to immunoassay-based validation

Urine α-fetoprotein and orosomucoid 1 as biomarkers of hepatitis B virus-associated hepatocellular carcinoma

Application of SELDI-TOF in N-glycopeptides profiling of the urine from patients with endometrial, ovarian and cervical cancer

Onco-proteogenomics identifies urinary S100A9 and GRN as potential combinatorial biomarkers for early diagnosis of hepatocellular carcinoma

Deciphering the peptidome of urine from ovarian cancer patients and healthy controls

Discovery and Validation of Novel Protein Biomarkers in Ovarian Cancer Patient Urine

Prediction of Chronic Kidney Disease Stage 3 by CKD273, a Urinary Proteomic Biomarker

A Urinary Fragment of Mucin-1 Subunit α Is a Novel Biomarker Associated With Renal Dysfunction in the General Population

Identification of Unique Blood and Urine Biomarkers in Influenza Virus and Staphylococcus aureus Co-infection: A Preliminary Study

Bladder cancer biomarker array to detect aberrant levels of proteins in urine

FGFR3 and Cyclin D3 as urine biomarkers of bladder cancer recurrence

Urinary Cysteine-Rich Protein 61 and Trefoil Factor 3 as Diagnostic Biomarkers for Colorectal Cancer

Clinical evaluation of urinary transforming growth factor-beta1 and serum alphafetoprotein as tumour markers of hepatocellular carcinoma

Urine Aquaporin 1 and Perilipin 2 Differentiate Renal Carcinomas from Other Imaged Renal Masses and Bladder and Prostate Cancer

An Improved Prediction Model for Ovarian Cancer Using Urinary Biomarkers and a Novel Validation Strategy

Identification of O-glycosylated Proteins That Are Aberrantly Excreted in the Urine of Patients with Early Stage Ovarian Cancer

Kidney Damage Biomarkers and Incident Chronic Kidney Disease During Blood Pressure Reduction

Tissue transcriptomedriven identification of epidermal growth factor as a chronic kidney disease biomarker

Associations of urinary epidermal growth factor and monocyte chemotactic protein-1 with kidney involvement in patients with diabetic kidney disease

Markers of early progressive renal decline in type 2 diabetes suggest different implications for etiological studies and prognostic tests development

Urinary epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors for rapid loss of renal function in type 2 diabetic patients with diabetic kidney disease

Urine AQP5 is a potential novel biomarker of diabetic nephropathy

Nonalbumin proteinuria is a simple and practical predictor of the progression of early-stage type 2 diabetic nephropathy

Infusion of autologous bone marrow derived mononuclear stem cells potentially reduces urinary markers in diabetic nephropathy

Urinary Biomarkers to Identify Autosomal Dominant Polycystic Kidney Disease Patients With a High Likelihood of Disease Progression

Urinary biomarkers are associated with incident cardiovascular disease, all-cause mortality and deterioration of kidney function in type 2 diabetic patients with microalbuminuria

Urine epidermal growth factor, monocyte chemoattractant protein-1 or their ratio as predictors of complete remission in primary glomerulonephritis

Urinary Tubular Injury Biomarkers Are Associated With ESRD and Death in the REGARDS Study

Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis

Highlights: • Urinary protein biomarkers are useful for diagnosis of many health conditions -kidney and cardio vascular diseases, cancers, diabetes, infections. • Liquid chromatography -mass spectroscopy is a powerful tool for urine proteomics, but used mostly in research labs. Many new biomarkers were discovered by this technique. • Immunoassays are widely used in both clinical and bio-analytical laboratories, • Infrared and Raman spectroscopies are promising tools for analysis of urine and di-agnostics due to relatively simple sample preparation, low-cost and short time of analysis. NO CONFLICT of INRTERST of any kind for the submitted manuscript Review