Determination of Cell Concentration by Using Optical Density Reading

Extraction Techniques and Applications: Biological/Medical and Environmental/Forensics

S. Damaraju , ... C.E. Cass , in Comprehensive Sampling and Sample Preparation, 2012

3.02.4.4 Stability of Isolated RNA over Extended Periods

Optical density (OD) measurements at ii wavelengths past spectrophotometer captured as a ratio (260/280 of >i.8) is also widely used to indicate RNA quality. An analysis of 44 chest cancer specimens (subset of 176 specimens described in a higher place for which both OD and RIN measurements were assessed) indicated that the samples that had RIN values of <half-dozen also showed relatively higher OD 260/280 ratios (1.89–2.0). Therefore, the reliability of OD measurements and comparing with the RIN measurements are valid but within a range (RIN of >6; only ane sample showed OD of 1.six with a RIN value of seven.i); lower RIN values and however higher or acceptable OD 260/280 ratios could potentially contribute to the poor quality of microarray information if tissue RNA integrity was solely assessed past OD measurements; these observations concur with those reported by others. ⁵⁵

Nosotros too tested the stability of RNA stored as a intermission in ethanol over extended periods (up to three years) at −80 °C (a subset of the samples analyzed by microarray). We tested 28 samples (RIN values ranging from half dozen to 9.7) for which RNA was extracted and quantified in 2003 (by OD 260/280 ratios) and these samples were once again processed for measurements past both OD and RIN in 2006. There was an excellent concordance between the OD measurements taken in the yr 2003 vs. 2006 (average OD of >ane.8). We also observed RIN values above vi.0 for 82% of these samples upon storage for over iii years in ethanol (Damaraju et al., information non shown). We conclude that storing isolated RNA in ethanol at −80 °C is a viable culling when dealing with rare samples, an approach that will facilitate sharing of the specimen as RNA instead of tumor between several independent investigators. Support for these observations likewise comes from independent studies, over again from a limited sample size (n = 27). ⁵⁶ RNA stored equally an ethanol precipitate was stable (OD, gel electrophoresis, and PCR-based tests) for up to 70 months as reported by independent research studies. ⁵⁶

Based on the above experiments and conclusions, we suggest that tumor banks routinely capture and comment specimens based on time of freezing/devitalization, cellularity of tumors, and should provide quality control for representative specimens. This is not a trivial task given the many challenges that pathologists face in a diagnostic laboratory. Nigh often, there are no dedicated pathologists for tumor banks and the support and contributions of clinical pathologists are disquisitional to the ability of a tumor depository financial institution to serve the inquiry community. Similarly, surgeon champions for research are invaluable given the priorities of patient care in busy operating rooms. In a clinical setting, the characteristics of the specimen are generated and put on the patient chart, but to routinely populate a tumor bank database with such data is expensive. Many banks capture clinical data fields simply after an investigator requests a sample set for research. While there are merits to both approaches, selecting cohorts is faster, has shorter turnaround times if these clinical characteristics are captured upfront. The CBCF Tumor Depository financial institution and participating pathologists contribute these information for every specimen going through a diagnostic laboratory. Detailed clinical characteristics are continuously updated in the CBCF Tumor Bank database forth with treatment information and patient outcomes (data non shown).

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Membrane Reactor

Ghasem D. Najafpour , in Biochemical Engineering and Biotechnology (Second Edition), 2015

16.4.2.four Analysis

The prison cell concentration was evaluated via optical density measurement based on developed calibration bend. The optical density was measured at 620 nm using a spectrophotometer (Unico, USA). Determination of glucose concentration was performed in nerveless 3 ml samples, and the prison cell from the sample was separated past micro centrifuge at 7000 g for 7 min (Hermle, model: Z 233 K-2 (Germany)). Finally, glucose concentration was determined past colorimetric method using DNS reagent. ^twenty Besides, ethanol concentration was measured using a gas chromatograph (Agilent, 7890A) equipped with a flame ionization detector (FID). The stainless steel packed cavalcade of 1.83 1000 length and two.1 mm I.D., 80/100 mesh Porapak Q (Supelco, U.s.) was used. The initial oven temperature was 120 °C held for i min. Then, oven temperature was programmed with a rate of 40 °C min^−ane until it reached 185 °C, and remained at the set temperature for 8.5 min. The detector temperature was 225 °C. Nitrogen as the carrier gas with a catamenia rate of 30 ml min⁻¹ was used. Likewise, 2-propanol or propionic acid (Merck, Federal republic of germany) was prepared in 10% (v/5) equally internal standard and added by a micropipette (Labnet, Frg), exactly 50 μl into 0.5 ml of each sample.

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Engineering Fundamentals of Biotechnology

J.H.T. Luong , ... M.B. Male , in Comprehensive Biotechnology (Second Edition), 2011

2.59.3.2.iii Online optical loss measurement

Equally described earlier, optical density and prison cell density readings can be related to biomass values, as OD readings in real fourth dimension and in the bioreactor can provide useful information for procedure control and optimization. In contrast to offline OD measurements using a visible wavelength (600–700   nm), inline OL-sensing using near-infrared wavelength (850   nm) is mainly dominated past scattering, as cells generally do non absorb strongly in the about-infrared region. At that place are two types of handful. Rayleigh scattering is indicated when the particles are much smaller than the wavelength of the calorie-free used in the measurement of optical loss. For handful by particles of size similar to or larger than the wavelength of the calorie-free, Mie scattering is applied. Because the sizes of bacteria (0.fifteen–ii   μm), eukaryotic cells (ten–30   μm), mammalian cells (10–100   μm), bubbles (100–25   000   μm), and detritus (0.1–100   μm) with respect to 600–850   nm low-cal, Mie handful must exist practical. In general, the relationship between OD and the cell measurement parameters (such as dry out jail cell weight) will be nonlinear. Therefore, a calibration run using the bodily medium is required to account for procedure variations. Once this relationship is established, the OD reading obtained online volition measure out and study the cell concentration with practiced precision and repeatability.

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Thermal Evaporation

Charles A. Bishop , in Vacuum Deposition onto Webs, Films and Foils (2d Edition), 2011

16.5.3 Errors in OD and Eddy Current Measurements Used for Coating Thickness

There has always been some question almost the truthful thickness of the aluminum coatings deposited. The aluminum coating will oxidize and the aluminum oxide is transparent as opposed to the opaque aluminum metal and and then the proportion of oxide will bear on the OD measurement. Similarly, the aluminum metal is conducting, but the aluminum oxide is an insulator so there will also exist an outcome on the eddy current resistivity measurement. This means that both the online measurement techniques are really measuring only the metal component of the coating thickness. As the oxidation of the metallic continues throughout the lifetime of the coating, the blanket thickness if measured by either of these two methods will also change with time. As the rate of oxidation tin can be affected by the nucleation and growth of the coating and hence the coating density and surface roughness, it is no surprise that using what appears to be an identical process on two different vacuum coating systems can produce coatings that are measured equally having ii different thickness coatings equally the oxide thickness may be unlike.

It is piece of cake to think of this oxide layer as being very thin, such as one–iii   nm, and to ignore it, simply as the aluminum coating thickness is as well sparse, the oxide tin represent a significant proportion of the total coating thickness. It was reported that measuring the OD in-line during metallization and immediately after venting the system, there could be a reduction of OD of more than xl% showing a significant amount of oxidation [22]. This high modify in OD is because the coating is and so thin. The oxide thickness that grows is similar irrespective of how thick the metal coating is, and and then for a coating of 10   nm or ten   µm, the oxide thickness later a twenty-four hours would be expected to exist of the order of i–3   nm on the surface of either blanket. In that location may exist small variations due to differences in crystal growth and surface roughness, just substantially the oxide growth will be similar. What was not considered at the time, but more recent work [23,24] has proven, is that the oxidation of the aluminum layer is on both sides of the aluminum metal as shown in the micrograph in Fig. sixteen.17. This micrograph clearly shows that the expected 1–3   nm surface oxidation is present at both the metal/polymer interface and the metallic/air surface. This micrograph shows the full oxide thickness to be shut to vii   nm range with a 29 nm thick aluminum coating giving an oxide thickness of the order of xx% of the total thickness. The implication of this is that to reach a particular OD for a customer, a much thicker coating needs to be applied in the metallizer to allow for the metal oxidizing over the fourth dimension before the customer receives the coating web and measures it.

Figure 16.17. Scanning transmission electron micrograph of an aluminum metallized coated polymer film showing oxide growth on both sides of the metal blanket [23].

TEM courtesy of Don McClure, 3M Corporate Inquiry Laboratory.

The longer the delay betwixt vacuum metallizing and the final use, the greater the difference in the measured OD during metallizing and the OD that volition exist present in the last production. Also, the higher the storage temperature and humidity, the greater the oxidation rate expected. As OD is used in the lite barrier calculations to decide the shelf life of products that tin can undergo photodegradation, it tin can be of import to know the charge per unit of oxidation to exist able to predict the final operation and prevent customer returns.

This detailed work [23,24] examined coatings after aging and produced new plots of the OD versus coating thickness for the metal and for the metallic with oxide layer included, as shown in Fig. sixteen.18.

Figure 16.18. Graph of aluminum blanket thickness versus optical density showing the theoretical performance from an optical model, the metal thickness, and the metal thickness consummate with oxide layers as measured using the scanning transmission electron microscope [24].

It is encouraging that the metal component of the thickness compares well to the theoretical optical model giving a confidence in the quality of the measurements. The graph as well shows the oxide thickness to exist similar for any thickness of aluminum coating.

The relationship betwixt the OD and resistivity was also plotted, and information technology was shown that the results can be fitted to the same curve irrespective of aging time, which, as both are affected by the loss of aluminum, might have been expected, and this is shown in Fig. 16.19. The regression of the data results in a polynomial curve fit where:

Effigy sixteen.19. Plot of optical density versus sail resistance for metallized sample aged five and 221 days [23].

Graph courtesy of GVE Ltd.

$Y=\mathrm{two.4425}{\mathrm{(}\mathrm{10}\mathrm{)}}^{-0.6535}{R}^2=0.9979$

with OD=Y and sheet resistance=X.

Using a combination of the above 2 graphs, it is now possible to plot both the OD and the sheet resistance against the equally-measured coating thickness onto polymer films. Using the bear witness of the oxide growth thickness, it is also possible to estimate the excess thickness of aluminum that needs to be deposited in order to deliver an optical or electrical resistance performance that tin meet customer requirements after information technology may have been in storage or transit for some weeks.

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Porous silicon for bacteria detection

N. Massad-Ivanir , Due east. Segal , in Porous Silicon for Biomedical Applications, 2014

12.3.1 Passive detection without capture probe – 'nonspecific' jail cell detection

In this approach, no receptors (e.thou., antibodies) were used in order to capture the target bacteria to the biosensor surface. Therefore, the spontaneous capture of the bacteria is accomplished by proper design of the transducer architecture and appropriate surface chemistry.

Sailor's grouping was the first to explore passive detection of bacteria and cell growth on PSi-based nanostructures (Alvarez et al., 2007; Schwartz et al., 2006). Alvarez et al. (2007) demonstrated a method for not-invasively monitoring the growth and Φ6 virus (a bacteriophage of the virus family Cystoviridae) infection of Pseudomonas syringae bacteria using a 1D PSi photonic crystal. The growth of the bacteria onto the PSi surface leads to an increased scattering efficiency, which was measured as a change in the intensity of the light reflected from the nanostructure (see Fig. 12.2a). The authors observed a linear human relationship between bacteria concentration and the optical betoken (see Fig. 12.2b). The detection limits were comparable to those obtained by optical density measurements. The main advantage of this cell-based biosensor set-up is that it allows bacteria cells to be monitored remotely without the need for sampling cells for plating or optical density measurements.

12.2. Effect of bacteria cell concentration on the peak intensity of 1D PSi photonic crystal. (a) The reflected scattering spectra from various concentrations of Pseudomonas syringae applied to a 1D PSi photonic crystal. (b) Intensity of scattered lite (wavelength = 591   nm) vs cell concentration.

(Source: Adapted with permission from Alvarez et al., 2007).

To date, all reported PSi-based optical biosensors consist of thin films of either mesopores (2–50   nm) or macropores (> 50   nm) (Naik and Ghosh, 2009; Vorontsov et al., 2007). These nanostructural sizes exclude biological species, such as bacteria cells and bacteria spores, from penetrating into the pores. Thus, 'direct' leaner capture may be monitored by intensity changes in the PSi reflectivity spectrum when bacteria cells reside on the acme surface of the porous nanostructure. While these sensing platforms may provide relatively high sensitivity and rapid response toward the target cells, they do not have advantage of the large porous volume.

Recently, a novel approach for extending the sensing capabilities of PSi for rapid detection of large biological species was introduced by Massad-Ivanir et al. (unpublished results) and Mirsky et al. (2013). A new class of two-dimensional (2nd) periodic macro-PSi array structures (MPSiAS), where the pore dimensions designed to fit the size of the target bacteria cells, are fabricated and applied equally an optical sensing platform for the detection of E. coli bacteria. In a close analogy to conventional reflective interferometric Fourier transform spectroscopy (RIFTS) sensors, the zero-guild diffraction of the reflected light presents a spectral interference pattern according to the phase accumulated inside the pores, assuasive for real-time detection of bacteria capture by measuring the variation in the constructive optical thickness (EOT) and the intensity of the sensor. The surface of the MPSiAS was chemically functionalized with positively charged amine groups. As most bacteria deport a cyberspace negative surface charge, adhesion of E. coli is promoted on positively charged surfaces (Gottenbos et al., 2001). The MPSiAS acts equally a lamellar (or a stage) grating that scatters the reflected light into a set up of diffraction orders at various angles co-ordinate to the human relationship between the periodicity of the grating and the optical wavelength. Fast Fourier transform (FFT) of the reflectivity spectrum from the MPSiAS layer provides a direct mode to monitor the EOT of the layer, which is characterized past a unmarried peak. Sensing is accomplished once leaner penetrate into the macropores, inducing measurable changes in the EOT that tin can be monitored and quantified in real time via RIFTS analysis. Thus, upon bacteria introduction (10⁵–10⁷ cell/mL) a rapid increase in EOT is observed, attributed to the entrapment of bacteria in the pores, leading to a refractive alphabetize increase. Current studies focus on immobilizing different bioreceptors such as antibodies and antimicrobial peptides onto the pore walls of MPSiAS to induce selectivity towards specific leaner and improve sensitivity.

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Stability and Stabilization of Biocatalysts

Guillán A. , ... Lema J.Chiliad. , in Progress in Biotechnology, 1998

ii.iii Belittling Methods

Glucose was determined past HPLC (Hewlett Packard, Palo Alto, USA) using an Aminex Ion-exclusion HPX-87H (Biorad, Richmond, CA) cation substitution column at 35°C with 5mM H₂SO₄ every bit mobile stage at a flow charge per unit 0.6   ml/min. The compounds were detected with a refractive index detector HP 1047A.

Free prison cell concentration was adamant by optical density measurements at 640   nm by expresed in dry out weight basis by using a calibration curve (1 absorbance unit ≅ 0.366g/l d.west.) For entrapped biomass, samples were obtained past dissolving 1   g of beads in 20   g/l tripotassium citrate solution.

The fraction of plasmid-containing cells (P⁺) in immobilized and costless cultures was analysed. To practise that, 1   thousand of gel beads were withdrawn from the culture and treated with tripotassium citrate solution until the beads were completely dissolved. The samples were diluted and spread on agar plates with selective and not-selective media. The plates were incubated at 30°C. Cells without the plasmid merely grow on plates with medium containing uracile, while cells containing the plasmid tin grow on both types of plates.

The assay for conclusion of β-l,3-glucanase activeness is based on the release of glucose from laminarin [18]. The assays were carried out by preparing reaction mixtures containing: 0. 25 % of laminarin, 0.65   ml of medium and acetate buffer up to 1   ml of total volume, giving 50   mM acetate buffer, pH 5.five. The corresponding substrate was added to a final concentration of 0.25%, and the reaction was performed by incubating at 37°C for 30   min. The reaction was stopped past maintaining the tubes for three   min in a boiling water bath. Residual glucose from fermentation broth was firstly removed by using PD-ten columns packed with Sephadex G-25 from Pharmacia Biotech (Uppsala, Sweden). The released glucose was determined by ways of an enzymatic-colorimetric method (GOD-PAD kit from Reactivos Spinreact S.A., Girona, Spain). Glucose is oxidized by glucose-oxidase to gluconate and hydrogen peroxide. After reaction, the absorbance was measured by spectrophotometry (Shimadzu, Kyoto, Japan) at 505   nm. 1 U of enzymatic activity was defined equally the corporeality of enzyme which released 1 μmol of glucose per hour.

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Specialty Polymers & Polymer Processing

Hiroyuki Sasabe , Tatsuo Wada , in Comprehensive Polymer Science and Supplements, 1989

6.3.4 Purple Membrane LB Films

Royal membrane (PM) is a plasma membrane of Halobacterium halobium and has an in-plane crystalline system of protein (bacteriorhodopsin; bR). ²⁶ The crystal structure of PM is a trimer of bR molecules within ane hexagonal unit prison cell (a  =   63   Å). Each molecule consists of approximately seven helical segments. bR molecules are localized in ca. 500   nm diameter discs in PM. bR is a low-cal-arresting protein and functions as a light-driven proton pump from the inside (cytoplasmic side) of the cell to its outside, which conveys the membrane potential. In lodge to obtain an ultrathin moving-picture show of PM, the Langmuir–Blodgett technique is quite useful. A pause of PM fragments in dimethyl-formamide of 25% concentration is spread on the surface of a subphase containing 0.two–0.iv   mM CaCl₂, and so compressed to form a monolayer and deposited on a substrate at 15–20 dyne cm^−one (1 dyne   =   10⁻⁵  N) by the horizontal transfer technique as shown in Figure x. ²⁷ The build up of films is checked past transfer ratio and optical density measurements. The morphology of the PM interface moving-picture show transferred onto a fleck of silicon wafer tin be straight observed past a scanning electron microscope (Hitachi S-900). The samples for photoelectric measurements are prepared onto ITO or Al electrodes. The counter Al electrode is deposited in vacuo to form an MIM jail cell.

Figure 10. Schematic representation of horizontal transfer technique for Langmuir–Blodgett films

Figure xi shows the typical photoresponses of 50 layers of purple membrane (PM) sandwiched with ITO and Al electrodes. The direction of photovoltaic electric current is from Al-top to ITO-bottom electrode of the jail cell, which is reasonable because the multilayer of PM is a z-type construction whose hydrophobic side faces the ITO substrate. It is well known that in PM the protons released from the end grouping of bacteriorhodopsin (bR), COOH, past photoexcitation motion across the membrane from inside (cytoplasmic side of PM fragment) to outside and hence the PM monolayer at the air/subphase interface statistically faces down its cytoplasmic side towards the subphase. ²⁷ The current and polarity of the dark current are dependent on relative humidity (RH), electrode materials (especially in the case of heteroelectrodes) and resistivity of the LB flick. The resistance of the ITO/PM/Al cell (expanse of electrode 0.28   cm²) is greater than 7   ×   10^xi Ω below 15% RH, and approximately ii   ×   x¹⁰ Ω at 70% RH.

Figure 11. Typical photoresponse of ITO/purple membrane LBF (50 layers)/Al cell under the illumination of a Xe lamp (75   Due west) (after Furuno et al. ²⁷ )

Both photovoltaic electric current and photovoltage at 1   s after excitation depend on the calorie-free intensity in a linear fashion, and their activity spectra are symbatic with the absorption spectrum of PM suspension. It is besides clear from crossed-illumination spectrophotometry that the photovoltaic response of the PM cell comes from the germination of M intermediate during the photochemical bicycle of bR. ²⁸

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NUCLEIC ACIDS | Immunoassays

J.M. WagesJr, in Encyclopedia of Analytical Science (Second Edition), 2005

Enzyme substrates

Considerable evolution has taken place in the field of new enzyme substrates, most notably the improver of several stable chemiluminescent substrates for alkaline phosphatase and peroxidase (Table three).

Table iii. Substrates for alkali metal phosphatase and horseradish peroxidase

Enzyme	Substrate	Product	Readout
Alkaline metal phosphatase	p-nitrophenyl phosphate (PNPP)	Soluble, yellow
	Nitroblue tetrazolium (NBT) in combination with v-bromo-4-chloro-3-indolylphosphate (BCIP)	Insoluble, purple
	iv-Methylumbelliferyl phosphate (4-MUP)	Fluorescent	358   nm excitation, 450   nm emission
	Fluorescein diphosphate	Fluorescent	490   nm excitation, 520   nm emission
	two′-[2-Benzothiazoyl]-6′-hydroxybenzothiazole phosphate [BBTP] (AttoPhos)	Fluorescent	435   nm excitation, 555   nm emission
	1,2-Dioxetanes (LumiPhos, CSPD)	chemiluminescent
Horseradish peroxidase	3-Amino-9-ethylcarbazole (AEC)	Insoluble, red
	iii,iii′-Diaminobenzidine (DAB)	Insoluble, brownish
	4-Chloro-1-naphthol	Insoluble, blue–blackness
	iii,3′,5,5′-Tetramethylbenzidine (TMB)	Soluble, xanthous	OD at 450   nm
	2,ii′-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)	Soluble, dark-green	OD at 410   nm and 650   nm
	o-phenylenediamine (OPD)	Soluble, brown	OD at 492   nm
	10-Acetyl-three-dihydroxyphenoxazine (Amplex Red)	Resorufin	OD at 563   nm; or 530   nm excitation, 590   nm emission
	Luminol, enhanced luminol	Chemiluminescent
	Lumigen PS substrates	Chemiluminescent

Colorimetric reagents generate a color change upon reaction with the reporter enzyme. Color changes are advantageous for rapid tests because they can exist detected visually. Precise quantitation or assay automation requires absorbance (optical density, OD) measurement, and a wide multifariousness of spectrophotometric and filter colorimeters are commercially available for this purpose. Despite the development of improved light emitting substrates, continued interest in colorimetric reagents is due to two factors. First, their cost is generally lower than the toll of more sensitive substrates. In particular, the price of instrumentation to read OD is low relative to fluorescence and chemiluminescence detectors. 2d, adequate sensitivity is achievable by adjusting either incubation fourth dimension of the enzymatic visualization reaction or the DNA amplification pace prior to hybridization analysis. Considering of its higher turnover number, peroxidase is the preferred label for colorimetric detection. Phosphatase assays will require longer incubation times, up to 1   h, whereas peroxidase assays are complete within 10   min. Although signal can be accumulated over fourth dimension to enhance weak signals, background also increases.

Fluorescent substrates are nonfluorescent until a quenching group is broken by the reporter enzyme, or enzyme action results in a shift in fluorescence emission. More often than not, fluorescence detection tin can be more sensitive than OD methods. Whether a theoretical increase in sensitivity is realized depends on the choice of enzyme and substrate besides as many other assay parameters.

Chemiluminescent substrates offer maximum sensitivity for reporter enzyme detection with a dynamic range of five orders of magnitude. Historically, their use has been limited by poor stability to heat and low-cal. Developments over the by ∼20 years, even so, accept yielded stable chemiluminescent substrates for alkaline phosphatase and beta-galactosidase. With these materials, less than 10⁻¹⁹  mol of alkaline phosphatase can be measured. Parallel evolution of peroxidase substrates has resulted in stable luminophores with sustained light yields to take advantage of the more rapid kinetics of the peroxidase reaction. The most developed of these reagents enables detection of peroxidase with similar detection limits and the same instrumentation as for the alkali metal phosphatase substrates. With glow-blazon reagents that produce sustained calorie-free output over many minutes, signal accumulation is possible by instrumental integration. Chemiluminescence readers are widely available in unmarried-tube and multiwell formats. Instrumentation can be programmed to read in either steady-land or kinetic mode.

Further enhancement in detection sensitivity of reporter enzymes is achievable by enzyme amplification or cascade reactions often termed 'enzyme cycling' assays. One arroyo is to use alkaline phosphatase as the reporter enzyme. Phosphatase cleavage of NADP forms NAD, which enters cyclic reactions catalyzed by alcohol dehydrogenase and diaphorase. Each turnover of phosphatase substrate initiates a cascade resulting in numerous detectable product molecules. Such approaches, perhaps incorporating chemiluminophores as the concluding product, agree hope for further extension of the sensitivity of immunoenzymatic methods.

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Present Status of Automation for Industrial Bioprocesses

R. Hausmann , ... B. Hitzmann , in Current Developments in Biotechnology and Bioengineering, 2017

25.ii.7 Quantification and Characterization of Biomass

If a biotechnical procedure uses living cells, the biomass is typically the virtually important variable for the procedure. Biomass is characterized by the total amount or concentration inside the bioreactor as well as its metabolic activity. In many applications, biomass is routinely quantified using different off-line methods, such as the measurement of optical density, gravimetric methods for the conclusion of cellular dry out weight, or microscopic methods. In addition to these off-line methods, in-line principles have been developed including turbidity sensors, impedance sensors, and microscopic methods.

In-line sensors for turbidity (optical density) measurements are based on manual, absorption, reflection, or light handful [two]. As a light source, lite-emitting diodes or lasers are commonly used, and adulterate lite is typically detected past photodiodes. For many processes, wavelengths between 840 and approximately 910   nm are used, because there is generally low absorption from the civilisation medium in the range. Even so, the optimum wavelength as well depends on the size of the cells, with college wavelengths used for larger cells, such as in mammalian cells cultures [thirteen].

Which measuring principle is applied is typically decided by depending on the intended awarding. Whereas the measurement of reflection is frequently chosen for high cell densities, scattering is mainly used for lower cell densities [14]. The determination of turbidity is highly influenced past other particles or air bubbling, and this organization does not let for the differentiation between feasible or dead cells [13]. Sensors for optical densities have been used successfully in the past for different biotechnical processes [15,16] with both microbial and bacterial cells.

The biomass in the bioreactor may also be adamant by measuring capacitance [17], which is used to notice intact cells, and which deed as capacitors in the liquid phase. In an alternate electrical field, their polarization and depolarization are measured, which can and so be correlated to the jail cell concentration. Ruptured cells are non polarized and therefore are non detected by this method; hence the capacitance measurement can be interpreted equally the viable cell concentration. This is a significant reward over turbidity measurements, which likewise detects cellular debris. However no details on metabolic country are provided by the measurements (e.1000., inactive cells). Capacitance measurements have been successfully used for the in-line conclusion of bacterial biomass [18] and mammalian prison cell cultures [19]. In improver, the evaluation of capacitative peaks over a range of electric field frequencies was used to estimate a jail cell size distribution for different microorganisms [20].

There are also several other electrical concepts for measuring biomass, biomass activity, or substrates/products, which are based on changes in electrical backdrop of either the medium or the cells, such as conductivity or permittivity [21]. Measurement of radiofrequency impedance was used successfully to determine full biomass concentrations, and it was likewise reported that impedance measurements may provide additional information past differentiating between growing or sporulating cells of Bacillus thuringiensis [22].

Another possibility for determining the concentration of cells is in situ microscopy coupled to automatized image-processing systems [23]. This technique has been employed successfully, east.g., to measure the mammalian cell culture density of Chinese hamster ovary cells [24].

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29th European Symposium on Calculator Aided Process Engineering science

Pui Ying Lee , ... Chi Wai Hui , in Computer Aided Chemical Technology, 2019

iii Effect

Performances of filtration is normally evaluated by the change in liquid concentration afterwards the process. In this study, the filtration efficiency is divers as the percent alter in optical density, which is proportional to the cell number density, from the original algal solution to the collected filtrate. The overall filtration efficiency represents the percentage change in jail cell number density of the solution as a whole without taking the specific change of cells at each size into consideration. Afterward the experiments and information assay, the overall filtration efficiencies of microalgae filtrations with different filter mediums are obtained. With the previously mentioned calibration curve, the cell number densities of the filtrate samples can be derived from the OD measurements. By plumbing fixtures the cell size distribution results into the proposed PBE model, the filtration coefficient ϕ defined in the model can exist derived.

As shown in Figure 1, the filtration efficiency is inversely proportional to pore size as expected and the relationship is observed to be almost linear. Although the pore size of from 15 to l um are manifestly larger than the cell size range of three to ix um, considerable amounts of cells are captured. Therefore, concerns are raised towards size selectivity of the microalgae filtration process and the mechanisms behind. With an original mean cell size of 5.381 um, Figure ii shows the mean filtrate jail cell size, which drops with decreasing filter pore size. From the pore size of 6 um to 15 um, it is observed that smaller mean pore size filters can finer capture smaller size cells since the cells cannot penetrate through the narrow pores. However, a drop of mean captured cell size is observed at 20 um pore size, implying that an increasing number of small-scale cells are captured even though the pores get larger. This data briefly indicates the tendencies of the filters to capture cells at dissimilar sizes.

Figure 1. Affect of filter pore size on the overall jail cell harvesting efficiency

Figure 2. Impact of filter pore size on the hateful filtrate cell size

Analysing size distributions of the captured cells tin can give additional information of microalgae filtration for studying the changes in mean cell size during the process. Figure three presents the size distributions of captured cells after the filtration experiments. It agrees with the previous findings that the number density of microalgae in the filtrate mostly decreases when a filter with smaller pores is used. Applying Population Balance Equation to simulate the filtration process, the harvesting efficiencies of microalgae cells at each size changing along with time and travelling altitude throughout the process are investigated. Subsequently numerical simulations of the developed PBE model with size distribution data from the experiments, the size specific filtration coefficients of microalgae filtered by medium of different pore sizes are obtained equally shown in Figure iv.

Figure 3. Size distributions of captured cells with dissimilar mean filter pore sizes

Figure iv. Derived size specific filtration coefficients of microalgae from the PBE model

Two unlike patterns are observed for smaller pore sizes (6, eight and 10 um) and larger pore sizes (15 and 20 um). For small pore sizes of 6, eight and 10 um, the coefficient increases with prison cell size, indicating that larger cells are more than favourable to exist captured. Among the iii curves, the 6um pore size curve has the steepest slope, which means it is more sensitive to cell size and its selectivity for large cells is higher. For large pore sizes of 15 and 20 um, convex curves are observed, indicating that both large and small cells are more favourable to be captured. The xx um pore size curve is comparatively steeper, such that the filter captures pocket-size and large cells ameliorate only oppositely information technology captures middle size cells worse than 15 um pore size filter.

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