INOM
EXAMENSARBETE TEKNIK, GRUNDNIVÅ, 15 HP
STOCKHOLM SVERIGE 2018 ,
Fotofysik av blod på en brottsplats
Mätning av enzymatisk aktivitet i blod PATRIC ELF
KTH
IN
DEGREE PROJECT TECHNOLOGY, FIRST CYCLE, 15 CREDITS
STOCKHOLM SWEDEN 2018 ,
Photophysics of blood on a crime scene
Measurement of enzyme activity in blood spots
PATRIC ELF
Photophysics of blood on a crime scene
Measurement of enzyme activity in blood spots Patric Elf
pelf@kth.se
SA114X Degree Project in Engineering Physics, First cycle Department of Applied Physics
Royal Institute of Technology (KTH) Supervisors:
Johnny Jussi, Dept of Applied Physics, KTH, Stockholm; RISE Acreo, Kista, Sweden Simon Dunne, Swedish National Forensic Centre, Link¨ oping
Examiner:
Ying Fu, Dept of Applied Physics, KTH, Stockholm
May 20, 2018
Abstract Svenska:
Syftet med den h¨ ar studien ¨ ar att analysera m¨ ojligheten till att ˚ aldersbest¨ amma blod med hj¨ alp av enzymer som blodet inneh˚ aller, f¨ or att se om dessa resultat i sin tur g˚ ar att anv¨ anda f¨ or att ge en uppskattning om hur l¨ ange sedan blodet l¨ amnade kroppen. Huvudsakliga anv¨ andningsomr˚ adet f¨ or det skulle vara vid en brottsplats, f¨ or att kunna ge ett tidsspann d˚ a ett brott har beg˚ atts.
Analysen best˚ ar av att ett serum som ¨ ar spikat med dessa enzymer har f˚ att torka ¨ over tid, och hur aktiviteten av enzymerna som ¨ ar beroende p˚ a enzymernas s¨ onderfall p˚ averkas. Problem som uppstod, s˚ asom intorkning och vad blod inneh˚ aller behandlas ocks˚ a, s˚ av¨ al som kort om hurvida det ¨ ar m¨ ojligt att konjugera kvant-prickar till enzymerna.
English:
The purpose of this study is to analyze the possibility to age-determine blood spots with the
help of enzymes which blood contains, in order to see if with these results it’s possible to give an
estimate of how long it has been since blood left the body. The main domain of this would be that
of a crime-scene in order to give a time-frame of when a crime has been commited. The analysis
consists of a serum that has been spiked with these enzymes gets to dry over time, and how the
activity of these enzymes, which are dependent on the enzymes denaturation change. Problems
which arose, such as drying of the blood, what blood contains, as well as a brief review of the
possibility to conjugate quantum dots to the enzymes are also reviewed.
Acknowledgements I want to thank...
Johnny Jussi for the patience and overall help with the the project, teaching me how assays work, how to pipette and much more. You have my sincerest gratitude for everything.
Ying Fu for allowing me to be part of this project, answering all of my silly questions and for being incredibly supportive and helpful in general.
Simon Dunne & Louise Elmlund for allowing me to be part of this project. I’m grateful for the feedback I got from our mail correspondence and phone-meetings.
RISE (Acreo) & Swedish National Forensic Centre for supplying the assay kits, en- zymes, laboratory equipment and more which made this project possible.
Tell me and I forget.
Teach me and I remember.
Involve me and I learn.
- Benjamin Franklin
Contents
1 Introduction 5
1.1 Scope and objective . . . . 5
1.2 Background . . . . 6
1.3 Blood . . . . 7
1.4 Enzymes . . . . 8
2 Materials and Method 9 2.1 Assays . . . . 9
2.2 Evaporation . . . . 11
2.3 Lactate Dehydrogenase . . . . 13
2.4 Alanine Aminotransferase . . . . 14
2.5 Aspartate Aminotransferase . . . . 14
2.6 Creatine Kinase . . . . 14
2.7 The plate readers and absorbance . . . . 15
2.8 Quantum dots . . . . 15
2.8.1 Quantum dot substrate conjugation . . . . 16
3 Results 17 3.1 Evaporation . . . . 17
3.2 Results from assays . . . . 20
3.2.1 LDH . . . . 20
3.2.2 ALT . . . . 24
3.2.3 AST . . . . 26
3.2.4 CK . . . . 28
4 Discussion and conclusion 31
Appendices 33
A Word list 33
B Disposable products used 33
C Measured activity in wells 34
D Protocol example: CK 35
1 Introduction
The importance of blood in modern forensic science can hardly be overstated. Blood is used to determine how wounds are inflicted through the analysis of blood-splatter patterns and to determine the identity of victims or suspects through the extraction and analysis of deoxyribonucleic acid (DNA), or more accurately the short tandem repeat deoxyribonucleic acid (STR-DNA), which are highly polymorphic tracts of repetitive DNA leading to a high genetic diversity.[1] The invention of both these techniques have revolutionized how forensic science is done.
Currently, to provide a time-frame of when a crime has been committed, things such as the body- temperature, state of the body, i.e. it’s decomposition, rigor mortis, and forensic entomology, the study of insects found on a crime-scene are used,[2] which are all well and good when there is a body to be found, otherwise forensic science have to rely on outside factors such as date and time indicators, mail, newspapers, expired and spoiled foods, stopped clocks etc.[3] This is of course a good way to get a rough estimate of when a crime took place, but there is a possibility that blood could give a higher accuracy of the time-frame.
Blood could prove to be a valuable source of such information because of its unique composition of proteins and low molecular weight compounds. Such an analysis would also improve the viability of evidence by being able to determine which blood-spots are relevant for the crime since there is no assurance that all blood-spots at a crime-scene are relevant. Some could be from days or even months before the actual crime was committed, which would remove the need to waste time and resources to perform DNA analysis on those samples. Due to the denaturation and various other changes of the compounds in blood, we should be able to use it to determine a time-frame of when the blood left the body.
1.1 Scope and objective
The purpose of this project is to investigate the possibility if we can use proteins present in blood to determine the time since it left the circulatory system. The project consists partially of a literature study in order to determine what has previously been done, assays of different enzymes and inves- tigating the possibilities of tagging enzymes with quantum dots in order to use their fluorescence to determine the concentration of the active enzymes in question. The hope is that this will help further studies in order to create a model, as well as a method to more easily determining the age of blood.
The literature study consisted of researching the topic and looking for studies that could be relevant to the topic and gain an understanding, which could be used as a basis for the project, and what kinds of results we would expect to find.
The assay-part is the main part of the project, in which we focused on four different enzymes which are all present in detectable amounts in blood, namely Lactate dehydrogenase (LDH), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Creatine Kinase (CK), of which we performed assays of all. Using multiple different enzymes provides improved information about the samples, as they react differently to environmental factors which can affect their rate of denaturation.
The Quantum Dot part of the project only involved theory about the possibilities of connecting
quantum dots to enzymes due to the time constraints of the project.
1.2 Background
The idea of using blood to get a time-frame of when a crime was committed is not a novel idea.
Various different ideas and techniques have been proposed over the years, dating as far back as to 1907 when Louis Tomellini from the university of Genoa developed a chart of 12 different figures to illustrate how the color of blood changes over a period of a year, or in 1930, when Schwarzacher attempted to find the relationship between bloods solubility in water and it’s age.[5] Various other ideas have been proposed and tested during the century since then, involving the use of blood or it’s components, such as the use of spectrophotometry analysis, electron paramagnetic resonance, near infrared spectroscopy and more.[5]
The main problem with all these techniques, and when dealing with blood in general, is that it is much easier to handle it in the laboratory with a controlled environment, while it is a completely different story out in the field. In the field things such as humidity, light and temperature change over time and play a crucial role in the changes that occur within the blood and it’s components.
Different kinds of spectroscopy techniques are common in life-science in general and when dealing with blood. A previous study which performed assays of CK and ALT found that they could find a correlation between the age of the enzymes and the absorbance of light. This was found to be due to the denaturation of the enzymes. Because of the different rates of denaturation, it was concluded that a single marker assay was not reliable enough to determine the age of a blood sample, but by using different enzymes or proteins with varying half lives, long and short respectively could provide a good result. They concluded that this method could be a viable way to determining the age of blood samples.[5]
Quantum dots have in recent years proven to be useful in bio-imaging and bio-sensing, because of
their high stability, brightness and small size. It is estimated to be 20 times brighter and 100 times
more stable than commonly used fluorescent markers, [6] and with the relative ease of coupling them
with proteins, which has seen improvements in recent years and happens frequently in life-sciences
such as in highly sensitive cellular imaging,[7] means that the use of quantum dots to determine protein
degradation in blood should prove effective.
1.3 Blood
Blood is the main circulatory body fluid, which has multiple functions, such as transporting nutrients and oxygen to the cells, removal of waste such as carbon dioxide and more. It accounts for roughly 7% of the total human body-weight, which means that on average a human carries with it 5 litres.[8]
Of these 5 litres, roughly 55% of the volume consists of plasma, which in turn is around 92%
water, 40-50% red blood cells and about 1% white blood cells, as in Figure 1.[9] The red blood cells are large microscopic cells that transport the oxygen and carbon dioxide through the body. It’s also they that give blood it’s characteristic red color, with the hemoglobin, which makes up 95% of a red blood cell.[9] Due to the sheer mass difference between these three components, it is easy to centrifuge blood in order to separate them. The simplicity in which these components of blood can be separated has made it common to use methods such as fluorescent spectroscopy on only the blood plasma, which is preferable, since the red color and dark tint of the red blood cells would interfere while looking at smaller components in the blood.
Figure 1: Schematic picture of cen- trifuged blood with the approximate distribution of the different layers.
Erythrocytes is another word for red blood cells.[10]
In blood, or more particularly, blood plasma, there is a huge array of different proteins and enzymes. These serve a variety of different functions, from transporting lipids, hormones and vitamins, acting in the immune system, to acting as enzymes in catalyzing different reactions.
All of these proteins and enzymes of course have different properties, and react differently to various factors, such as heat, light, being in different kinds of solutions, which all affects their various half lives and denaturation-processes. But overall, it is safe to say that all of them, as with all bio-molecules, degrade over time, and that heat speeds up the degradation due to it speeding up the biological processes.
In eukaryotic cells there are mainly two major pathways of protein degradation, which speed up this natural process in or- der to regulate the amount of a certain protein which should be present at any given moment, the ubiquitin-proteasome path- way and lysosomal proteolysis,[11] but since we are focusing on blood samples which are not in cells, or more importantly, pro- teins which are in plasma, these sources of degradation should not affect the overall denaturation of our proteins, and only the basic half lives should be of importance, which are dependant on factors which are more controllable, such as temperature.
One important thing to note, is that according to one study[12], the act of drying blood, does not significantly af- fect the overall degradation and detectability of the proteins present, with the average correlation being estimated at 0.970,
using the non-parametric rank based Spearman method between wet and dried samples. It also found that the detectability of some proteins was neither affected by storage over long periods of time (three decades) in cool conditions either. This is significant, since the blood-spots on crime-scenes are more often than not at least partially dried up before the forensic teams can arrive at the scene. In some cases, it can take days or even weeks before a crime-scene is discovered. It is also significant for the reason that it means that samples containing enzymes can be stored in cool conditions in order to preserve integrity.
While working with components that are found in blood, it is common to use a human serum.[13]
A serum in this regard is the blood plasma without the fibrinogens. The fibrinogens are clotting
agents, which help the blood to coagulate.
1.4 Enzymes
Figure 2: Schematic picture of an enzyme converting a substrate into two products.
An enzyme is an organic catalyst. This means that they accelerate chemical reactions. Most enzymes are proteins and many proteins which are present in blood are enzymes that catalyze different reac- tions in order to regulate the concentrations of various compounds in the blood. The molecules which the enzyme acts upon are called sub- strates, which in the process are converted into products. The most important fact about enzymes is, as with all catalysts, that they do not get consumed in the reaction, but rather remain the same in the same state. Enzymes also don’t alter the equilibrium of the reaction.
This means, that after an enzyme has catalyzed a reaction, as seen in Figure 2, it will then be able to catalyze another substrate.
It is worth noting that the admixture of enzymes in the blood varies greatly depending on a lot of different factors such as the sex of the individual, age, physical fitness and more, so the concentration of the enzymes can’t directly be used to determine the age of a blood spot, but rather it’s the rate of denaturation of the enzymes that is of interest. This rate varies depending on which enzyme it is but also depending on which isoenzyme it is, with some enzymes such as Lactate Dehydrogenase (LDH) having a half-life of 30 to 162 minutes in chickens varying depending on the isoenzyme.[14] It was important to keep the half-life in mind while working with enzymes, and that different isoenzymes can have different half lives, since as an example, there would be negligible amounts left after a week with an enzyme that has a half-life of 30 minutes, and hence it’s rate of denaturation would be more relevant to measure over a shorter time-span.
In enzyme chemistry, a regularly used unit is the ”Enzyme unit”, U. The definition of U is the amount of the enzyme that produces a certain amount of enzymatic activity, which means the amount that is catalyzed in the conversion of 1 µmole of the substrate per minute.
For this to be relevant, the conditions must also be specified, such as the temperature, pH and concentration. In the assays which we per- formed, which is reviewed further in the Section 2.1, we used activity
assay kits with the only factor that we had to consider which would vary from the specifications was
the temperature. The temperature used for those assays was room temperature (20-22 ◦ C).
2 Materials and Method
2.1 Assays
Assays of four different enzymes which are present in blood were performed, consisting of Lactate De- hydrogenase (LDH), Aspartate Aminotransferase (AST), Creatine Kinase (CK) and Alanine Amino- transferase (ALT). These were all done with activity assay kits ordered from Sigma-Aldrich or Abcam, in which for each assay we spiked a new HS sample with one of the enzymes. The spiked HS samples were, as well as unspiked HS in others, deposited and left to naturally dry by the environment over different periods of time in in the wells of a 96-well plates. The unspiked HS acted as a negative control, in which no enzymatic activity was to be expected as the active enzyme was not present. A problem that was faced was that these activity assay kits were not designed to be used with dried samples, and as this study focuses on dried blood spots we had a problem with evaporation, which is reviewed further in Section 2.2.
When spiking HS with any of the enzymes, the goal was to mimic normal levels in human blood, or plasma which serum more accurately resembles. Normal levels of our four enzymes in adults bloodstream are:
• LDH 100-190 U/L[15]
• ALT 7-56 U/L[16]
• AST 10-40 U/L[16]
• CK 22-198 U/L[17]
We tried to mimic these values to the best of our abilities while spiking the HS used for the assays by using a value somewhere within the normal range. For each measurement the used HS was spiked with only one of these so there would be no interference from the other enzymes.
Assay activity kits were used for these assays. They all included Assay Buffer, Substrates, Stan- dards and Positive Control.
• An Assay Buffer is a solution which is resistant to changes in pH. It serves to stabilize the desired pH during the assays and act as a medium which the other components can be mixed into.
• Substrates are simply the substrates for the reactions which the enzymes catalyze.
• A Standard in this context is a sample with a known concentration of the particular product which is detected, and by depositing it in different amounts, it gives a so called standard curve which can be used to evaluate how much of the product that has been produced in our spiked HS sample wells. The standards varied depending on the enzymes, as the different enzymes catalyze different reactions and produce different products. The standard for the different enzymes are NADH for LDH, Glutamate for AST, NADH for CK and Pyruvate for ALT.
• A Positive Control gives a large amount of enzyme activity, which serves as a control to compare to the spiked samples in order so see that the reaction in the samples happens as intended and is detectable.
• Some of them also included an Enzyme Mix, which is mix of auxiliary enzymes, as well as a Developer, which is a chemical agent which helps develop the assay.
• ALT, AST and CK also included probes, which react with the enzyme mix to form an inter-
mediate, which reduces a colorless Probe to a colored product with strong absorbance at the
wavelength specified.
The substrates as well as the enzyme mix, developer and probes for the enzymes where they were required was mixed into a master reaction mix with assay buffer. When the master reaction mix is added to the enzyme samples, the enzymes immediately begin to work on catalyzing their respective reactions.
When spiked HS had been deposited into wells for the second to last time point, meaning when there was an hour left until we want to run the assay, the assay activity kits components were prepared.
The basic procedure was as follows:
• An hour before the assay was about to be put into the plate reader the assay activity kits components, the positive control, the standard and the master reaction mix was prepared.
• The last samples, the positive control and the standard were put into the wells.
• All wells were filled up with buffer to the amount specified in the protocol. It was important to have all of the wells at the exact same volume in order to get comparable measurements, hence we had to calculate for the evaporation. The calculations of the specific amount needed to compensate for evaporation can be found Section 2.2.
• The master reaction mix was added to all wells which activates the biocatalytic cascade.
• The well plate was put into the plate reader, which is further described in Section 2.7, which illuminated the samples with light of a specific wavelength and continual optical measurements were taken, by measuring the absorbance at the set wavelength that was instructed for each of the activity assay kits. This wavelength varied, depending on what kind of probe was used and what type of reaction that was taking place.
All our assays were performed in triplicates, to minimize potential problems caused by errors, such as problems with pipetting, bubbles in the plate wells or other unforeseen events. The basic plate layout that we found most appropriate, as can be seen in Table 1, gave us enough wells in order to both deposit each required constituent, as well as discard wells from our calculations that might be erroneous without altering the results. The spiking of the enzymes to HS was not exact, as small errors could occur, and as such we could not know which volume that would give a readable result.
Because of this, we reserved two columns of the 96 well plate, as can be seen in Table 1, to test for this.
It is important to note that for the test wells, we simply added different volumes of spiked HS, i.e.
different concentrations, to the wells, and tested it versus the standards in order to find an appropriate response. The Activity Assay Kits, except for the LDH Activity Assay Kit only had components for 100 assays, which meant that it was important to get it right as it meant that only one well plate could be filled up with one kit.
It was important to store everything correctly during these assays as the degradation of enzymes
depend on temperature, and cold or frozen samples, as most biomolecules, degrade slower at lower
temperatures. For this reason, everything that is not immediately used has to be stored in a freezer
at -20 ◦ C until it was about to be used, and then thawed using a water bath. The spiked HS and
negative control were however stored in a fridge at 4 ◦ C to remove the need to thaw and freeze them
multiple times to prevent damage.
Table 1: The plate setup used in most of our LDH, ALT, AST and CK assays, where PC is Positive Control wells, NC is Negative Control wells, standard is the standard wells, test wells is where we test for the appropriate volume, and sample is where we put our samples.
The basic procedure for all of these assays are the same, as described above, with minor differences.
Such as the preparation of the different enzymes, where LDH and ALT are kept in an ammonium sulfate suspension, and CK and AST as a lyophilized powder. In the ammonium sulfate suspension case, the first step is to transfer an appropriate amount of the suspension to a small vial with a needle, the vial is next centrifuged for as long a time is needed in order to cause a pellet to form in the bottom of the vial, after discarding the supernatant the pellet is resuspended in Phosphate-Buffered Saline (PBS) and HS is spiked according to the desired concentrations. For the lyophilized powder, it depends on the enzyme, AST can directly be resuspended in PBS and spiked to HS. CK on the other hand, first requires it to be resuspended in a Glycyl-Glycine suspension before it is possible to spike HS.
In order to spike the HS with the appropriate amount we used the formula:
2ml · Y = x · C, (1)
Where x is the volume PBS with the enzyme dissolved, C is the amount of the enzyme in the PBS in U/L, and Y is the desired amount in U/L. We used 2ml since that was generally more than enough for all the used wells on the plate.
Due to these differences a protocol was made before each assay which was easy to follow, one of which is added as Appendix D to this report. With each of the activity assay kits, there also came protocols, but as they contained a lot more information than was needed for the immediate work so it was deemed more efficient to have a simple step-by-step protocol to work with.
2.2 Evaporation
Since the samples used in the assays were left to dry over time and the assay kits are originally intended to be used with liquid samples, a problem that was faced was that of evaporation. Hence the rate of evaporation was needed in order to determine how much buffer was needed to be added to the different wells to have the right volume in the well while doing the coloriometric measurements.
To get a baseline for different amounts of human serum, different concentrations were deposited into small vials, and left opened. The weight was then measured periodically over 8 hours. This is not an ideal way to measure the evaporation, mainly since the vials were slightly deeper than the wells on the plates, but since evaporation depends on so many different factors, such as temperature, surface area of the droplet and airflow[18], it was however a good indicator and did give a decent estimation, especially considering that the surface area would be difficult to determine. We expect that the main source of evaporation should be water.
As seen in Figure 3, it was determined with this method for a vial deposited with 10µL older than
8 hours, all of the HS is expected to have dried out. This evaporation rate is however not going to be
the same in the wells in well plates, as the depth and dimensions of the well are different, but it did
show that this is a viable, although crude way, to determine how much buffer should be added. We
did however notice a quite large variance in evaporation between the different samples.
0 1 2 3 4 5 6 7 8
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035
Weight [Grams]
10 L average, R
2=0.98232 20 L average, R
2=0.98951 30 L average, R
2=0.98362
Figure 3: Weight of different amounts HS over 8 hours. Each plot is the average of 3 different samples with R 2 for a first degree polynomial.
Due to the uncertainty of these results, especially considering that the height of the wells are
shorter than that of the vials used and the fact that we shouldn’t see that kind of variance in evapo-
ration between the samples the accuracy of the scale came into question. It was decided to redo the
evaporation-tests with another scale, (a Kern Analytical Balance, ABS 120-4N, Z741077-1EA) as well
as by performing another test, by measuring evaporation with human serum in different containers
and substrates as can be seen in Figure 4; Cardboard coffee cups; Wood from ice cream sticks; Micro-
scope plate slides; Wells cut from a 96 well plate, which gives the exact same dimensions as wells for
the assay experiment. This was also done so that we can repeat this with human blood to be more
confident that HS is a good substitute for human blood.
Figure 4: Mug, Ice cream stick, microscope glass slide and cut well used in the evaporation experi- ments. The same setup was used with blood.
2.3 Lactate Dehydrogenase
Lactate dehydrogenase is as the name implies, a dehydrogenase enzyme. That means that it is an enzyme which is a oxidoreductase enzyme which catalyzes the transfer of electrons from one molecule to another. In this case it means catalyzes the interconversion of pyruvate and lactate by reducing NAD + to NADH, and also oxidizing NADH to NAD + in reverse as seen in Figure 5.
Figure 5: Interconversion of pyrovate and lactate, catalyzed by LDH. [19]
In our assays, LDH reduces NAD + to NADH, which is specifically detectable by colorimetric assay at 450nm[20], which by measuring how this absorbance changes over time, we can determine the activity of the LDH, which in turn should be proportional to the amount of LDH present in the sample.
In the body LDH is released from cells into the bloodstream after tissue damage or red blood cell
hemolysis, and since LDH is a reasonably stable enzyme it is used to evaluate the presence of damage
and toxicity in tissue and cells.[20] Normal LDH levels in an adults bloodstream range from 100-190
U/L while in a newborns it is normal with levels between 160-450 U/L[15] and hence, it is prevalent
and should be a great candidate to measure it’s degradation over time. In this LDH assay we used
150 U/L.
2.4 Alanine Aminotransferase
Alanine Aminotransferase is a transaminase enzyme, meaning it catalyzes a transamination between a amino acid, in our case L-alanine to a keto acid, in our case ketoglutarate, as seen in figure 6.
Figure 6: ALT catalyzes the transfer of an amino group from L-alanine to ketoglutarate, producing pyruvate and glutamate. This process is reversible. [21]
The ALT Activity kit contains a flourescent peroxidase substrate, with which we can determine the ALT activity from the resulting coupled enzyme assay, since we get a colorimetric (570 nm) product[22], which is proportional to the pyruvate generated.
Normal ALT levels in range between 7 to 56 units per liter, which is lower than that of LDH, but still enough in order to be detectable in our assay with the probe. ALT is found primarily in liver or serum, but occurs elsewhere in other tissue as well. Increased ALT serum levels are used as a marker for liver injury.[22]
As we got unsatisfactory results in our tests with concentrations within the normal range, with a very low enzymatic activity, we had to use 300 U/L and 600 U/L in our ALT assay.
2.5 Aspartate Aminotransferase
Aspartate Aminotransferase, also commonly known as serum glutamic oxaloacetic transaminase is as the name suggest a transaminase enzyme. It catalyses the interconversion of an amino group from aspartate to glutamate as can be seen in figure 7. The normal range of AST is between 10-40 U/L and mildly elevated levels considered when they are about 2-3 times that of in the normal range.[23]
Hence 30 U/L was decided for the concentration in our HS to use in the assay. AST is more broadly distributed in tissue, and a rise in AST levels occur in case of a variety of diseases or in injuries of multiple different tissues, such as the skeleton and the heart.[24]
Figure 7: AST catalyzing the interconversion of aspartate and ketoglutarate to oxaloacetate and glutamate, as done in our assay.[25]
2.6 Creatine Kinase
Creatine Kinase, as found in human blood, converts creatine into phosphocreatine and ADP using
ATP as seen in figure 8. CK is a common enzyme in the cells and in the blood it’s concentration
varies greatly, from 22-198 U/L according to some sources. CK is commonly used in blood tests as
a marker to detect damage in CK-rich tissue as it is released in case of damage. It is prevalent in
striated muscles, such as the heart of the brain.[26]
Figure 8: Creatine Kinase converting creatine into phosphocreatine.[27]
In the CK assay we used 100 U/L.
2.7 The plate readers and absorbance
The plate reader works by measuring the absorbance of each of the samples in a 96-well plate, by shining a light of a specific set frequency through the sample and measuring how much of the light that passes through and how much is absorbed. The values of the absorbance received is arbitrary from the plate reader and cannot be related directly to the concentration of the marker but must be compared to a standard plot.
A standard plot is generated by depositing different known amounts of a known substance, in essence, NADH, glutamate or pyruvate for LDH, ALT and AST respectively and then plotting a curve for the absorbance generated for the different concentrations. We see that the absorbance dependance is linear for the amount of the set standard, and hence we can calculate how much of that standard that is generated in the sample wells. In actuality we had a standard with a known concentration, and deposited it in different amounts in the standard wells as can be seen in Table 1.
When calculating the standard plot, we use R 2 to determine the goodness of fit for the points to the plot. In essence, it is the proportion of the variance from a fitted line that can be predicted from the measured values. In general, if the R 2 is above 0.96, it’s considered a good fit.
2.8 Quantum dots
Quantum dots, in essence, are tiny semiconductor particles which generally are between 2-10 nanome- ters in size and possess controllable optical properties. These particles have proven useful in a wide array of fields, such as in generating sharper images for televisions and in-cell imaging due to their small size.[28][29]
The color of the quantum dots are generally dependant on the size and shape of the dot, as larger
QDs tend to emit a longer wavelength, while smaller ones emit shorter wavelengths. Due to the
specific wavelength emitted, it is easy to distinguish different QDs from each other, as seen in figure
9, where it can be done with the naked eye by irradiating them with UV-light. With fluorescence
spectroscopy, it is possible to have numerous different QDs in a sample and distinguish them, and
they are easy to activate by simply irradiating them with low-intensity light.
Figure 9: Quantum dots in solution, irradiated with UV-light.
2.8.1 Quantum dot substrate conjugation
There are anti-bodies, or substrates, for nearly every imaginable protein. Thus, by conjugating a QD to one of these substrates, it would prove a great way to mark said proteins.
As with everything else, QDs obey the laws of chemistry. The carboxyl-group of biomolecules,
in this case a substrate, can be coupled to amino-coated QDs by the help of EDC. Carbodiimide
conjugation would work by activating a carboxyl-group for reaction with the amine via amide-bond
formation.[30] There is however a problem with this idea, since there might be sheer mechanical issues,
because of the size of the enzymes and quantum dots, they would block the substrate from attaching
to the enzyme. This can however be solved by attaching a peptide or linker between the QD and the
substrate, and hence, removing the mechanical obstacle.
3 Results
3.1 Evaporation
As we can see in Figure 10, the evaporation time varies greatly depending on the surface. Both the wooden ice cream sticks and paper cups weight started to increase during the test. This was likely due to the absorption of water from the air due to the humidity. The plots for the evaporation on an ice cream stick and on a paper cup are thus corrected for this by the weight difference from our blank (one without any HS) being removed from the rest. Due to the fact that not everything had evaporated after 8 hours in the plate well where 50 µL had been deposited, we decided to place a well on highly accurate scale overnight with a camera capturing it’s weight every 60 minutes. As can be seen in Figure 11b, about 10 percent of the weight is left when it is all dried up. We can also determine that the evaporation is higher in a well than in a vial, with about 3 µL evaporating per hour for a 10 µL well as seen in table 2. For this plot with the different volumes we used first degree polyfits in MATLAB, which by calculating the R 2 from the measured values we can determine are good. It’s also interesting to note that the evaporation seem to slightly slow down with smaller volumes.
0 1 2 3 4 5 6 7 8
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(a)
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(b)
0 0.5 1 1.5 2 2.5 3 3.5
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(c)
0 0.5 1 1.5 2 2.5 3
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(d)
Figure 10: Weight of different amounts HS over time, where each plot is the average of three different samples.
(a) Well cut from a 96-well plate, (b) Paper Cup, corrected for weight increase of the cup, (c) Wood from ice
cream stick, corrected for weight increase of the wood, (d) Miroscope glass slide.
0 1 2 3 4 5 6 7 8 Time [Hours]
-1 0 1 2 3 4 5 6 7 8
Weight [Grams]
10
-3Cut plate well Paper cup
Wood from ice cream lolly Microscope glass slide
(a)
0 2 4 6 8 10 12 14 16
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L average 30 L average 50 L average 50 L long time well
(b)
Figure 11: During weighing of HS, (a) The measured weight difference from initial weight of blanks (containers and substrates without samples), (b) Weight over time in well plate, with one 50µL well over a long period of time.
Volume/Degree First degree polynomial fit R 2
50 µL 0.0497-0.0040x 0.99970
30 µL 0.0284-0.0036x 0.99951
10 µL 0.0100-0.0032x 0.99846
Table 2: The polynomial fit for the different volumes, in grams. Polyfits for 50 µL use 8 hours, 30 µL use 6 hours and 10 µL use 2 hours.
Next, we redid the previous measurements with whole blood instead of HS. As can be seen in Figure 12 compared to Figure 10, we notice a slight difference in evaporation between HS and blood, mainly on wood. This is thought to be mainly because the wood didn’t absorb the blood as well as it did with HS as can be seen in Figure 12c, but rather it remained as a droplet on top, which meant that the overall surface area of which the blood could evaporate from didn’t increase as it did with the HS.
In a plate well however, the evaporation does seem to be similar, as can be seen when comparing
Figure 11b and Figure 13b.
0 1 2 3 4 5 6 7 8 Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(a)
0 0.5 1 1.5 2 2.5 3 3.5 4
Time [Hours]
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
Weight [Grams]
10 L 30 L 50 L
(b)
0 0.5 1 1.5 2 2.5 3 3.5
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(c)
0 0.5 1 1.5 2 2.5 3
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L 30 L 50 L
(d)
Figure 12: Weight of different amounts human blood over time, where each plot is the average of three
different samples. (a) Well cut from a 96-well plate, (b) Paper Cup, (c) Wood from ice cream stick, (d)
Miroscope glass slide.
0 1 2 3 4 5 6 7 8 Time [Hours]
0 0.5 1 1.5 2 2.5 3 3.5 4
Weight [Grams]
10
-3Cut plate well Paper cup
Wood from ice cream lolly Microscope glass slide
(a)
0 2 4 6 8 10 12 14 16
Time [Hours]
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Weight [Grams]
10 L average 30 L average 50 L average 50 L long time well
(b)
Figure 13: During weighing of human blood, (a) The measured weight difference from initial weight of blanks (containers and substrates without samples), (b) Weight over time in well plate, with one 50µL well over a long period of time.
Substrate/Volume HS, 10 µL HS, 30 µL HS, 50 µL Blood, 10 µL Blood, 30 µL Blood, 50 µL
Well 3 8 13 3 8 12
Cup 1.5 2.5 3.5 2 2.5 3.5
Wood 1 2 3 0.5 2 3
Glass 0.5 1 1 0.5 1 2
Table 3: Estimated time in hours for how long it takes for a spot to completely evaporate.
The results from our trials with evaporation were as would have been expected, with around 90%
evaporation for the HS as seen in Figure 11b, which is roughly the amount of water that it contains.
And as water was to be expected to be the main source of evaporation. It is interesting to note how vast the dependence of the surface the evaporation actually is, with the depth of a well significantly increasing the evaporation-rate of HS. It was surprising how similar the rate blood evaporated. The main difference seemed to be that it was not absorbed into surfaces such as wood.
3.2 Results from assays
We did plates with assays for each of the four enzymes multiple times. In the results we however only review one plate for each as it is representative for that enzyme.
When the spiked HS for the assays were not in use the vial of spiked serum was stored in a cool fridge at 4 ◦ C to prevent degradation.
Corrections for negative control means that the average value for the negative controls for each of the set time points is subtracted from the sample wells same time points. All assays were done at 20-22 ◦ C.
3.2.1 LDH
We found that there indeed was a change over time for our LDH samples, but more surprising was
the significant impact of the background absorbance as seen in Figure 14. The HS used in this assay
was spiked with LDH in order to be 150 U/L, which is within the normal range in humans.
0 5 10 15 20 25 30 35 40 Time [Minutes]
0 0.5 1 1.5 2 2.5 3
Absorbance [450nm]
LDH 0h sample well, corrected for negative LDH 0h sample well
Average for Negative 0h wells Most active standard well Positive Control
Figure 14: Example 5 µL 0 hour old sample wells, both corrected for the background absorbance and not corrected, as well as the average with the most active NADH-standard well and positive control.
It is interesting to note how the negative control changes over time.
These measured values cannot tell us the concentration directly, since the scale of the absorbance
is arbitrary, we used the NADH-standard plot in order to determine the amount of NADH generated
by the LDH. The standard was run in triplicate to minimize the possibility of bad wells making the
whole assay useless. As seen in Figure 15, one of the 10 µL wells shows significantly lower values than
the other two, thus indicating something has gone wrong during the preparations. Some deviations
between the triplicates are natural, but such a large deviations as we are seeing here is just not within
the margins of error - thus it is discarded and removed from calculations in order to improve the
accuracy of our results.
0 5 10 15 20 25 30 35 40 Time [Minutes]
-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Absorbance [450nm]
0 L 2 L 4 L 6 L 8 L 10 L
Figure 15: NADH standard wells as they were in the plate reader. The concentration of the NADH- Standard was 1.25 mM. Notice how the different volumes match up and the absorbance linearly increasing dependant on volume, except for one of the 10 µL.
With the results from the standard well, it was possible to calculate a polynomial fit for the absorbance to the nMole NADH generated as seen in Figure 16 using MATLAB.
With this fit, we could calculate the LDH Activity (U) with the simple formula:
LDHActivity = B · SDF
RT · SV (2)
Where B = Amount (nmole) of NADH generated between the initial and penultimate reading before the most active sample wells absorbance crosses that of the highest standard well, which is to be considered the final read. SDF is the Sample Dilution factor, which is 1 since the sample is not diluted, RT is the reaction time (final read minus initial read), and SV is the Sample Volume in mL.
The initial read was to be done after 2-3 minutes after adding the reaction mix, which was about the
time it took to start and setup the plate reader. It is interesting to note that the LDH activity, as seen
in the plot in Figure 17, does not immediately slow down, but rather increase for a few hours. This
might be due to issues, such as that the older samples don’t completely dissolve immediately when
reconstituting the samples before the read by the plate reader when buffer and master reaction mix
is added, meaning that there remains a dried spot in the bottom of the well. It is important to note
the high degree of variance that we see in the activity, as seen in Figure 17 in the standard deviation
in the error bars.
0 5 10 15 NADH [nmole]
-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
Absorbance [450nm]
Fitted curve, y=0.1111x+0.0022, R 2 =0.99797 Corrected measured NADH standard wells
Figure 16: Fitted NADH Standard curve. Notice the high degree of accuracy, of R 2 being above 0.99.
The faulty 10 µL standard well was removed from these calculations. y = 0.11111x+0.0022.
0 10 20 30 40 50 60 70
Age of samples [Hours]
20 40 60 80 100 120 140
LDH Activity [mU/mL]
5 L NC
Figure 17: Plot over LDH Activity, as well as the activity for the negative control. The LDH activity
is corrected for the negative control (NC).
3.2.2 ALT
For ALT we had inconclusive results of what would be the best concentration of enzyme. In earlier attempts we tried concentrations at 30 U/L, which were in the within normal range of ALT in human blood, but those proved to be too low, since they never crossed the Pyruvate Standard. So we decided to spike the human serum to a total concentration of 300 U/L and 600 U/L, and then deposit 10 µL samples in the plate wells. As could be expected, the higher concentration gave us a faster response as can be seen in Figure 18.
We got a slight spread in the Pyruvate-Standard for the different volumes as can be seen in Figure 19a, but since the R 2 value we got from out linear fit is still over 0.98 it is still considered acceptable, as correlation is still high.
00:00:00 00:05:00 00:10:00 00:15:00 00:20:00 00:25:00 00:30:00 00:35:00 00:40:00 00:45:00 00:50:00 00:55:00 01:00:00 01:05:00 Time [HH:MM:SS]
-0.5 0 0.5 1 1.5 2 2.5
Absorbance [450nm]
600 U/L ALT, corrected 600 U/L ALT 300 U/L ALT, corrected 300 U/L ALT Average NC Most active standard PC
Figure 18: Example wells for 600 U/L and 300 U/L, with and without correction for the negative
control, as well as negative control, the most active standard and a positive control.
00:00:00 00:15:00 00:30:00 00:45:00 01:00:00 Time [HH:MM:SS]
-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
Absorbance (570nm)
0 L 2 L 4 L 6 L 8 L 10 L
(a)
0 2 4 6 8 10 12
Pyruvate [nMole]
-0.5 0 0.5 1 1.5 2
Absorbance [570nm]
Fitted curve: y= 0.16424x--0.0659,R
2=0.98062 Corrected measured NADH standard wells
(b)
Figure 19: plots of the Pyruvate Standards. (a) Pyruvate Standards, corrected for the background by subtracting the average for the 0 µL standard wells. (b) The standard plot for Pyruvate.
The ALT activity was calculated in the same manner as for LDH with the formula ALT Activity = B · SDF
RT · SV (3)
With B is the amount of Pyruvate generated over the reaction time, SDF is 1, SV is 10 µL. In order to calculate the reaction time RT we have to look at Figure 18, we have an initial lag phase until around T init =10 minutes for both samples before we see a linear rise in absorbance, while T end =20 for the 600 U/L sample and T end =25 for the 300 U/L sample until they cross the most active standard.
We notice, as expected, that the higher concentration generates a higher ALT activity, and that they degrade at the same rate. What was strange however is that it was such low activity.
As can be seen in Figure 20, most of the drop in activity occurs during the first 10 hours, and after that it remains almost static.
-1 0 1 2 3 4 5 6 7 8 9 10
Age of samples [Hours]
35 40 45 50 55 60 65 70
ALT Activity [milliunits/mL]
300 miliunit/mL 600 miliunit/mL
(a)
0 10 20 30 40 50 60 70 80 90 100
Age of samples [Hours]
35 40 45 50 55 60 65 70
ALT Activity [milliunits/mL]
300 miliunit/mL 600 miliunit/mL
(b)
Figure 20: plots of ALT Activity over time. (a) 10 hour plot for the average ALT Activity with error
bars. (b) 100 hour plot for the average ALT Activity with error bars.
3.2.3 AST
In our AST Assay we pipetted 10 µL of 30 U/L AST spiked Human serum in our wells. For our AST assay we had multiple issues, such as our Glutamate-standard as can be seen in Figure 21a. These erratic plots can possibly be explained by some problem while depositing the samples. This in turn yielded a R 2 of 0.94 when calculating the plot for the average of each standard volume. This R 2 is lower compared to the other assays, which does affect the reliability of the activity.
We also had a problem with the machine shutting down in the middle of the read, but we were able to restart it after some time, hence the missing time points in Figure 22.
The absolute values for the AST Activity isn’t the most important thing in these assays however, since what we’re interested in is in finding the decrease over time, which should remain the same regardless.
0 50 100 150
Time [Minutes]
-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbance [450nm]
0 L 2 L 4 L 6 L 8 L 10 L
(a)
0 2 4 6 8 10 12
nMole Glutemate 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Absorbance [450nm]
Fitted curve: y= 0.057667x-0.062889,R
2=0.94157 Corrected measured NADH standard wells
(b)
Figure 21: plots of Glutamate Standards. (a) Glutamate Standards, corrected for the background.
(b) Glutamate Standard plot for average of triplicate of standards.
0 50 100 150 Time [Minutes]
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbance [450nm]
AST sample well, corrected for neg AST sample well
Average Negative Control Most active Standard Positive Control
Figure 22: Absorbance for AST sample well, both which was corrected for the egative control and wasn’t, the negative control, most active standard well nd positive control.
0 10 20 30 40 50 60 70 80 90
Age of sample [Hours]
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
AST Activity [milliunits/mL]
10 L
Figure 23: AST Activity plot.
Another issue that was faced was that the concentration which we spiked the serum with was too
low. That can be seen in Figure 22 as they never reach the highest Glutamate Standard. But in any
case, it still has it’s rise-phase, even though it’s quite long, which is usable, which we can plot the
average AST activity, as seen in Figure 23. The AST activity, is as usual calculated as AST Activity = B · SDF
RT · SV (4)
with B is the Glutamate generated during the reaction time RT, which in this case is the full interval, with the sample volume SV=10 µL and SDF = 1. It is important to note that we spiked a 1h well and a 0h well of negative control during the test in order to see if anything was actually happening due to it’s slow rise as can be seen in Table 7 in Appendix C, but those values were omitted during the calculations, such as correcting for the background.
Unfortunately the activity for our AST samples, and the change in activity proved too low to be able to draw any clear conclusions from these results.
3.2.4 CK
The CK assay was performed with 10 µL samples, with 100 U/L CK spiked in the human serum.
During this assay we also had some minor issues with the standard wells, mainly that the wrong volume was added, so we had a span from 0 µL to 12 µL instead of 0 µL to 10 µL. This however didn’t cause problems since we still got a usable standard plot, with a good R 2 as can be seen in Figure 24. In this assay as well we got quite low results, probably due to a low concentration as can be seen in Figure 25.
0 5 10 15 20 25 30 35 40
Time [Minutes]
-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbance [450nm]
0 L 2 L 4 L 6 L 8 L 10 L 12 L
(a)
0 5 10 15
nMole NADH -0.4
-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbance [450nm]
Fitted curve: y= 0.10028x--0.23146,R
2=0.99262 Corrected measured NADH standard wells
(b)
Figure 24: (a) Absorbance for CK standard wells. (b) CK Activity plot.
0 10 20 30 40 50 60 Time [Minutes]
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Absorbance [450nm]
